Structure of Silk studied with NMR

Progress in Nuclear Magnetic Resonance Spectroscopy 39 (2001) 301±352

www.elsevier.com/locate/pnmrs

Structure of Silk studied with NMR Chenhua Zhao, Tetsuo Asakura* Department of Biotechnology, Tokyo University of Agriculture and Technology, Koganei, Tokyo 184-8588, Japan Accepted 4 August 2001

Contents 1. 2. 3. 4. 5.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The amino acid composition and primary structure of silk . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Isotope labeling of silk ®broin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Solution structure of silk ®broin by solution NMR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Use of chemical shifts and chemical shift contour maps for conformational analysis of silk ®broin in the solid state . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6. Determination of torsion angles for oriented silk ®ber by solid state NMR . . . . . . . . . . . . . . . . . . . . . 7. Determination of torsion angles for silk by spin-diffusion NMR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8. Determination of the atomic distance of silk model peptides using REDOR . . . . . . . . . . . . . . . . . . . . 9. Determination of Silk I (B. mori silk structure before spinning in the solid state) by combination of several solid state NMR methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10. Dynamics of silk ®broin from NMR relaxation measurement and 2H NMR . . . . . . . . . . . . . . . . . . . . 11. Silk ®broin synthesis in silkworm monitored by in vivo NMR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1. Introduction Silks are generally de®ned as spun ®brous protein polymer secretions produced by biological systems [1,2]. Silks are synthesized by a variety of organisms including silkworms (and most other Lepidoptera larvae), spiders, scorpions, mites and ¯ies. Silkworm silk (Bombyx mori silk) has been the most intensively studied and is synthesized in specialized sets of modi®ed salivary glands and extruded from spinnerets * Corresponding author. Fax: 181-423-83-7733. E-mail address: [email protected] (T. Asakura).

301 304 308 310 313 319 328 333 335 341 347 349 349

located in the head of the larva [3]. The majority of silks are spun into air, although some aquatic insects produce silks with differing compositions that are spun under water. Recently, much attention has been paid to silk from textile engineers to polymer chemists and biomedical scientists. For example, silk ®broins from silkworms or spiders can produce strong and stiff ®bers at room temperature and from an aqueous solution, whereas synthetic materials with comparable properties must be processed at higher temperatures and/or from less benign solvents. Potential applications range from biomedical devices to bullet-proof vests and

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Fig. 1. Life cycle of B. mori.

Fig. 2. Silk gland of B. mori larva.

parachutes. Nowadays, it is possible to produce new silk-like materials with bacterial methods for the expression of silk proteins, or to synthesize large quantities of silk protein with mammalian host `factories'. The atomic level information on the silk structure gives us the answer to why the silk ®bers have such excellent properties. X-ray and electron diffraction methods together with infrared spectroscopy have been applied for this purpose. However, silks are semi-crystalline ®brous proteins and the primary structure is essentially very heterogeneous although there are many repeated sequences locally. Thus a new analytical technique is required for their full investigation. NMR can cover from solution state to solid state including the gel state, and even the liquid silk in a living silkworm. In addition, atomic level structure can be obtained by using mainly solid-state NMR techniques developed recently and by combining them with the stable isotope labeling of silks and their sequential model peptides. Our purpose is to review the structure of several silk ®broins determined mainly by NMR as well as to describe the NMR analytical method.

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Table 1 Amino acid compositions of silk ®broin from B. mori, S. c. ricini, A. pernyi and A. yamamai, and of the silk sericin from B. mori (mol%) Amino acids

B. mori ®broin

B. mori sericin

S. c. ricini ®broin

A. pernyi ®broin

A. yamamai ®broin

Gly Ala Ser Tyr Asp Arg His Glu Lys Val Leu Ile Phe Pro Thr Met Cys Trp

42.9 30 12.2 4.8 1.9 0.5 0.2 1.4 0.4 2.5 0.6 0.6 0.7 0.5 0.9 0.1 Trace ±

13.5 5.8 34 3.6 14.6 3.1 1.4 6.2 3.5 2.9 0.7 0.7 0.4 0.6 8.8 0.1 0.1 ±

33.2 48.4 5.5 4.5 2.7 1.7 1 0.7 0.2 0.4 0.3 0.4 0.2 0.4 0.5 Trace Trace 0.3

26.7 48.1 9.1 4.1 4.2 2.9 0.8 0.8 0.2 0.7 0.3 0.4 0.3 0.3 0.5 Trace Trace 0.6

26.1 48.1 9 3.9 4.5 3.5 0.8 0.7 0.1 0.7 0.3 0.4 0.2 0.4 0.6 Trace Trace 0.7

Let us describe Bombyx mori silk has been the most intensively studied. The life cycle of B. mori is summarized in Fig. 1 [4]. In about 50 days, it completes its life cycle of four different metamorphosing phases: egg or embryo, larva, pupa and adult (moth). Of the life cycle, about half is the larval stage, the only stage at which they consume food, in the form of mulberry leaves. Pupation occurs at the

Fig. 3. Schematic texture of silk thread.

end of spinning (or cocoon formation); the latter takes 3±4 days. Thus, silkworm silk is produced primarily at one stage in the life cycle, during the ®fth larval instar just before the molt to the pupa. The gland in which the silk of B. mori is secreted is shown in Fig. 2 [3], and consists of three relatively distinct regions. Fibroin, the main component of silk proteins, is exclusively synthesized in the posterior region of the silk gland and is transferred by peristalsis into the middle region of the gland in which it is stored as a very viscous aqueous solution until required for spinning. In the walls of the middle region of the gland, another silk protein, sericin is produced which coats the silk ®broin, acting as an adhesive; both proteins have unique and easily distinguishable amino acid composition (Table 1). The two glands join together immediately before the spinnerets through the anterior region, and then the ®ber are spun into the air. In the extruded thread, the two ®broins cores remain distinct (Fig. 3). B. mori silk ®broin can assume two distinct structures in the solid state, namely silk I before spinning, and silk II after spinning: silk ®ber. The corresponding structures have been investigated by X-ray ®ber diffraction [2,5,6,12±18], electron diffraction [16,19], conformational energy calculations [20,21], infrared spectroscopy [2,17,22,23], and 13C and 15N

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cross-polarization magic angle spinning (CP/MAS) NMR [7±11,17,24±26]. Despite a long history of studying silk I, its structure determination was dif®cult because attempts to induce a macroscopic orientation of the sample for X-ray diffraction, electron diffraction or solid state NMR, readily cause a conversion of the silk I form to the silk II form [7,17,27±30]. Recently, we have resolved the molecular conformation of silk I as a repeated bturn type II, using solid state NMR methods such as 2D spin-diffusion NMR under off magic angle spinning, Rotational Echo DOuble Resonance (REDOR), and using 13C CP/MAS chemical shift data [31]. Concerning the structure of silk II, Marsh et al. [12] were the ®rst to propose an anti-parallel b-sheet model based on a ®ber diffraction study of native B. mori silk ®broin ®ber. Fraser et al. showed that the polypeptide sequence (AGSGAG)n exhibits a slightly greater inter-sheet spacing than (AG)n, but in accordance with the anti-parallel b-sheet model [2]. Here A is alanine, G glycine and S serine. Subsequently it was pointed out by Lotz et al. that (AG)n in the silk II form must possess some intrinsic structural disorder, because the inter-sheet G±G and A±A distances are increased and decreased, respectively, when compared to polyglycine and polyalanine, as determined by X-ray and electron diffraction [19]. Recently, Takahashi et al. [18] reported a more detailed X-ray ®ber diffraction analysis of B. mori silk ®broin based on 35 quantitative intensities. Having analyzed four types of models for the silk II form in terms of the experimentally derived R factor, they proposed that two anti-polar anti-parallel b-sheet structures are statistically stacked with different orientations, occupying the crystal site with a ratio of 1:2 [18]. Even though the local protein conformation is still the basic b-sheet as proposed by Marsh et al. the re®ned silk II model accounts for the stacking of the b-sheet planes in two different arrangements. X-ray and electron diffraction methods are powerful approaches to obtain structural information on the crystalline regions of the silk ®ber, which predominantly consist of the repeated sequence (AGSGAG)n and which can be reliably modeled by the polypeptide (AG)n. However, diffraction methods cannot yield any results on the amorphous domains of silk II, in which the repeated AG sequences are known to

contain interspersed Tyr(Y) and Val(V) residues. Solid-state NMR spectroscopy, on the other hand, provides direct structural information about individual amino acid sites, especially when coupled with stable isotope labeling of B. mori silk ®broin and synthetic model peptides. Thus, the structural information from both crystalline and amorphous domains can be obtained. As mentioned above, much attention has been paid to silk. The tensile strength and yield at fracture of these natural silk ®bers are comparable to those of synthetically produced ®bers, such as Kevlar [3]. Because of the exceptional mechanical properties of silk ®ber, the ®ber is believed to be a composite of interconnected crystalline (b-sheets) and amorphous regions [32,33]. The b-sheet crystals are believed to be responsible for the high strength and stiffness of the ®ber, while the elasticity of the ®ber arises from the amorphous regions. A number of NMR techniques have been applied to elucidate the structure and dynamics of silk ®broin in our laboratory. The combined use of isotopically labeled samples and NMR techniques has also been extremely advantageous in elucidating the silk structure, dynamics and also biosynthetic pathways [34±36]. The majority of the NMR studies have been performed on ®broin and ®bers produced by the domestic silkworm, B. mori, and to a lesser extent on ®broin produced by the wild silkworms, Samia cynthia ricini (S. c. ricini) and Antheraea pernyi (A. pernyi). While the focus of this review is on NMR studies of silk ®broin and ®bers from these silkworms, a section on NMR studies of spider silk is also included to present a more complete picture of NMR research on silk. 2. The amino acid composition and primary structure of silk In the chemical composition of ®broin from B. mori as summarized in Table 1, the sum of G and A contents in the silk ®broin is 73 mol% and other amino acids are S12.2 mol%, Y4.8 mol%, and V2.5 mol% [3,37], four of them are G, A, S and Y, which makes up approximately 90 mol%. Thus the character of the silk ®broin is originally due to the presence of G and A residues. From a colloidal aqueous ®broin solution, about 55% of the ®broin

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Fig. 4. Primary structure of B. mori silk ®broin [42].

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Fig. 5. Primary structure of A. pernyi silk ®broin [43].

can be cleaved by enzymatic hydrolysis with chymotrypsin and isolated as crystalline powder suitable for X-ray studies [38]. It consists of a polypeptide in which G, A and S occur in the molar ratio of 3:2:1.

The silk protein from B. mori silkworm is known and has a primary structure consisting largely of a repeating sequence of six amino acid residues such as (GAGAGS)n [39±40].

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Fig. 6. Comparison of 50.3 MHz 13C solution NMR spectra of the carbonyl region among (A) non-labeled (natural abundance), (B) [1- 13C]Alalabeled, (C) [1- 13C]Gly-labeled, and (D) [ 15N]Gly-labeled silk ®broins of B. mori in aqueous solution at 408C. The peaks were labeled from a to p with increasing magnetic ®eld in Spectrum A and assigned in Fig. 12 [9].

The complete sequence of B. mori silk ®broin gene has been determined by Mita et al. [41] and Zhou et al. [42] by means of combining a shotgun sequencing strategy with physical map-based sequencing procedures. According to Zhou et al. the deduced ®broin amino acid sequence is 5263 residues long, with a molecular weight of 391,367 Da. The B. mori silk ®broin gene consists of two exons (67 and 15,650 base pair, respectively) and one intron (971 base pair). The ®broin coding sequence presents a spectacular organization, with a highly repetitive and Gly-rich (,45%) core ¯anked by non-repetitive 5 0 and 3 0 ends. The repetitive core is composed of 12 repetitive domains (R01±R12) separated by 11 amorphous domains (A01±A11) as shown in Fig. 4. The sequences of the amorphous domains are evolutionarily conserved and the repetitive domains differ from

each other in length by a variety of tandem repeats of sub-domains of ,208 base pair which are reminiscent of repetitive nucleosome organization. Most of the GX dipeptide units where X is Ala in 65%, Ser in 23% and Tyr in 10% of the repeats are present as part of the two hexapeptides GAGAGS (432 copies) and GAGAGY (120 copies) [42]. A 1350 base pair nucleotide sequence from the Chinese oak silkworm, A. pernyi has been determined [43]. Fig. 5 summarizes the complete sequence of A. pernyi ®broin. The deduced amino acid sequence was partitioned into thirteen polyalanine-containing repetitive motifs, which are characteristic of A. pernyi ®broins. Eleven of these arrays can be classi®ed into two types of motifs depending on the difference in amino acid sequences following polyalanine. Repetitive motifs structurally similar to those in A. pernyi

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Fig. 7. 9 MHz 15N solution NMR spectra of (a) non-labeled silk ®broin (natural abundance), (b) [ 15N]Ala silk ®broin obtained by oral administration of [ 15N]Ala to silkworm, and (c) [ 15N]Ala silk ®broin obtained by the rotation culture procedure (amplitude was reduced compared with other two spectra) [49].

were detected in S. c. ricini and a homologue of Japanese oak silkworm, A. yamamai. Spiders make their webs and perform a wide range of tasks with up to seven different types of silk ®ber. These different ®bers allow a comparison of structure with function, because each silk has distinct mechanical properties and is composed of peptide modules that confer different properties. An examination of the cDNAs and genes of the spider silks sequenced to date shows that all silks are chains of iterated peptide motifs [44±48]. The consensus sequences for the repeating peptides of major and minor ampullate silks and ¯agelliform silk from N. clavipes are repeated many times throughout the length of each protein. The small peptide motifs can be grouped into four categories: (1) GPGXX/GPGQQ; (2) (GA)n/An; (3) GGA; (4) spacers. Dragline silk has a tensile strength comparable to that of Kevlar coupled with a reasonable elasticity and is therefore an extremely strong ®ber.

3. Isotope labeling of silk ®broin Selective isotope labeling of silk ®broin is required in the NMR experiments to obtain site-speci®c structural information and high S/N ratio. Two kinds of isotope labeling methods have been used: oral administration of isotope-labeled amino acid to silkworm or in vitro organ culture of the posterior silk glands. The former labeling is achieved biosynthetically through the use of an arti®cial diet supplemented with the isotope-labeled amino acids or by injection of the amino acids as an aqueous solution during the ®fth instar larval stages. The isotope-labeled and oriented silk ®ber can be easily obtained from the cocoon. Fig. 6 shows the expanded carbonyl region in the solution 13 C NMR spectra of naturally abundant silk ®broin (A), [1- 13C]Ala (B) and [1- 13C]Gly (C) labeled silk ®broins [9]. The labeling ratio was high for both isotope-labeled silk ®broin samples and can be used

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Fig. 8. Possible metabolic pathway of how 2H2O injected into the silkworm is incorporated into the alanine methyl group [50].

for solid-state NMR experiments. The presence of Ala±Gly and Ser±Gly sequences is con®rmed by the observation of the Ala and Ser carbonyl peaks coupled with 15N Gly nuclei (D). The in vitro organ culture method was also possible for the isotope labeling because the posterior silk glands that produce the silk ®broin are immersed in hemolymph directly in silkworm. Therefore, the isotope-labeled amino acid in the medium is incorporated effectively into the silk ®broin. Actually, a large amount of the silk ®broin can be produced continuously and effectively by culture of the posterior silk gland and the isotope-labeled silk ®broin could be obtained in this way. Fig. 7 shows 15N solution NMR spectra of B. mori silk ®broins [49]. Fig. 7(a) is the spectrum of silk ®broin in the natural abundance (non-labeled) state. Fig. 7(b) is 15N labeled Ala silk ®broin obtained by oral administration of 15N Ala to silkworm. Although glutamic acid or aspartic acid is added to the arti®cial diet containing 15N Ala, in order

to avoid the transmutation from Ala to these amino acids, the 15N Ala labeling of the sample was still low. Fig. 7(c) is the 15N spectrum of 15N Ala labeled silk ®broins obtained by the cultivation of silk gland in the medium containing 15N Ala. The 15N enrichment of Ala residues was 20 times the natural abundance 15N concentration, which was suf®ciently high for 15N solid-state NMR analysis of the silk ®broin structure. Similarly, 2H-labeling of silk ®broin ®bers was also achieved by the oral administration method and several kinds of 2H labeled silk ®broins were obtained for solid state 2H NMR analysis [50]. During the labeling process, an interesting metabolic pathway was discovered: a highly 2H-labeled sample of the methyl group in Ala is obtained by using only the 2 H2O oral administration method. The possible metabolic pathway from 2H2O into the alanine methyl group is illustrated in Fig. 8 [50], where 2H2O incorporation by the catalysis of fumarate hydratase, glycolysis, which involves pyruvate, takes place in the

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Fig. 9. 50.3 MHz 13C solution NMR spectral changes of the liquid silk extracted from the mature larva of S. c. ricini as a function of urea concentration. The peaks marked h are attributable to the a-helical peak and the peaks marked with asterisks are attributable to the random coil peak [55].

cytosol of cells, whereas the TCA cycle occurs within the mitochondria. 4. Solution structure of silk ®broin by solution NMR The structure of B. mori silk ®broin in aqueous solution or in the middle silk gland is important in connection with the mechanism of the formation of ®broin silk. A random coil structure has been established in dilute aqueous solution [51], but considerable confusion exists concerning the conformation in concentrated aqueous solutions or when the protein is stored in the middle silk gland of the silkworm. An intramolecular b -type has been proposed by Iizuka et al. [52] using light scattering, intrinsic viscosity, ¯ow birefringence, circular dichroism (CD), optical rotatory dispersion (ORD), and infrared methods and also using the small-angle X-ray scattering data reported by Kratky [53]. However, a recent CD study shows

the occurrence of a `helical' conformation at concentration higher than 5 w/v% [54]. In order to check this, the 13C solution NMR spectra of S. c. ricini and B. mori silk ®broins have been compared. The conformation of S. c. ricini silk ®broin was analyzed on the basis of the `doublet peak' observations of the Ca, Cb and carbonyl carbons of the Ala residue, which were assigned to a-helix and random coil conformations [55] (Fig. 9). These peaks were assigned by observing the denaturing effects of urea on liquid silk extracted from the mature larva of S. c. ricini. With increasing urea concentration, the high®eld component of the Ala Cb peak, (peak h), shifts to lower ®eld, and the low ®eld peaks (peak h) of the Ala Ca and CyO shift to higher ®eld. Other peaks marked by asterisks and the Gly Ca peak do not give observable shifts. The carbon resonance of the Ala residue coalesce in 8 M urea solution, suggesting that the peaks marked by h arise from the a-helix conformation and those marked by an asterisk arise from a random coil conformation. Thus, two conformations,

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Fig. 10. 50.3 MHz 13C solution NMR spectra of S. c. ricini silk ®broin in the alanine carbonyl region observed as a function of temperature and the calculated spectra for the poly (l-alanine) sequence as functions of the statistical weight w and the parameter A. The peak marked with h is attributable to the a-helical peak (Fast exchange between a-helix and random coil of poly (l-alanine) sequence). The peak h p marked with asterisk is the end group of poly (l-alanine) sequence and the peak, c, attributable to the random coil peak [57].

random coil and a-helix, coexist in aqueous solution of S. c. ricini silk ®broin [56]. The helicity of each residue of the sequence of the Ala residues in S. c. ricini silk ®broin protein was calculated by using the Bixon± Scheraga±Lifson theory for the helix±coil transition of poly (l-alanine) including the hydrophobic sidechain interaction [57]. The helix±coil transition of S. c. ricini silk ®broin is also induced by changes in temperature, and 13C NMR spectroscopy is able to monitor the conformational change as shown in Fig. 10. The peaks h and h p in the carbonyl resonance region show gradual up-®eld shifts as the temperature was increased. This behavior can be interpreted as resulting from the decrease of the helicity due to a decrease in the statistical weight of the helix state w [57].

Without any external forces, we can compare the solution structure of silk ®broin stored in the silk gland directly between two kinds of silkworms, B. mori and S. c. ricini using in vivo NMR. As shown in Fig. 11, the peaks from silk ®broins (A and B) are easily identi®ed because the silk ®broin should be deleted in pupa (C). The chemical shifts of the sharp Ala Ca, Cb and CyO peaks of the silk ®broin in B. mori silkworm (A) are in agreement with those of the random coil of silk ®broin in S. c. ricini silkworm (B) [55]. Thus, in contrast to the CD data, 13C NMR clearly rules out the presence of a-helical portions in the B. mori silk ®broin [58]. The 13C solution NMR spectrum of B. mori silk ®broin is shown in Fig. 12 [51]. The signals in the

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Fig. 11. 50.3 MHz 13C solution NMR spectra of the silk gland portion of both intact S. c. ricini (a) and B. mori (c) mature larvae and of the abdomen of S. c. ricini pupa (b) [4].

spectrum are very sharp in spite of the high molecular weight, indicating very fast segmental motion of the silk ®broin chain. The amino acid composition determined from the solution NMR is in agreement with that from amino acid analysis. Sixteen peaks were observed in the expanded carbonyl region [9]. These peaks were assigned to the primary sequence at the penta-peptide level from biosynthetic labelling of the carbonyl carbons and from the change in the spectrum after chymotrysin hydrolysis (Fig. 12). Fig. 13 shows the 1H-coupled and decoupled 13C spectra of the carbonyl carbon for the Gly residue of [1- 13C]Gly-labeled ®broin [58]. The observation of

the long-range coupling constants between 13C and 1 H nuclei is expected to yield information concerning the local conformation around the N±C a bond of the speci®ed residue of B. mori silk ®broin. Splitting of the peaks in the carbonyl region of the 1H-decoupled spectrum was assigned to speci®c sequences [9]. The value of the 3JC 0 ±N±Ca±H was readily determined from the spacing of the doublet for each Gly carbonyl carbon peak in the 1H-coupled spectra. After correction by considering the peak-to-trough ratio of the doublets, the determined 3JC 0 ±N±Ca±H values were 2.8 Hz for the G±A±G±S±G sequence, 2.4 Hz for the G±S±G±A±G sequence and 2.6 Hz for the G±A±G±A±G sequence at 1.8 w/v%. These values

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Fig. 12. 50.3 MHz 13C solution NMR spectrum of B. mori silk ®broin in aqueous solution (8.7 w/v%). The carbonyl resonance region was expanded and assigned by penta-peptide sequence level [9,51].

decrease slightly with increasing concentration; 2.5 Hz for the G±A±G±S±G sequence, 2.0 Hz for the G±S±G±A±G sequence and 2.2 Hz for the G± A±G±A±G sequence at 7.3 w/v%. The C 0 ±N±Ca± H torsion angle f has a value of 2608 for the Ala residue with silk I structure [31], and so 3JC 0 ±N±Ca±H is calculated to be 20.75 Hz. This indicates that silk I conformation of the silk ®broin might appear with increasing concentration. Fig. 14 shows the partially relaxed 13C NMR spectra of the silk gland portion of intact B. mori silkworm, which was obtained to get the spin±lattice relaxation time T1. In spite of the presence of two kinds of silk proteins i.e., silk ®broin and sericin, in the middle silk gland, the plots are essentially single exponential [58]. The results indicate that only one component is present in the liquid silk (concentration < 30 w/v%), from the viewpoint of dynamics, and the mean correlation time for the segmental motion in such liquid silk was determined

to be 2.2 £ 10 210 s, which is longer than the corresponding value, 1 £ 10 210 s, in dilute aqueous solution (2.1 w/v%) [51]. 5. Use of chemical shifts and chemical shift contour maps for conformational analysis of silk ®broin in the solid state The 13C chemical shifts of the amino acid residues of polypeptides and proteins are signi®cantly dependent on the local conformation (as de®ned by the torsion angles, f and c ) of the particular residue, as well as on the manner of hydrogen bonding. Therefore, it is possible to determine the conformation (such as right-handed a-helix, b-sheet, v -helix, 31helix, and collagen-like triple helix) of particular regions of a polypeptide or protein, by examining the 13C NMR spectra of these samples.

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Fig. 13. 1H-coupled 50.3 MHz 13C solution NMR of the Gly carbonyl region of [1- 13C]Gly-labeled B. mori silk ®broin with increasing concentration in 2H2O at 408C. Sample concentration (w/v%): 7.3 (B), 5.5 (C), 4.4 (D), 2.2 (E), 1.8 (F). 1H-decoupled spectrum (A) of [1- 13C]Gly-labeled B. mori silk ®broin [58].

In the analysis of the conformation of B. mori silk ®broin in the solid-state, a number of such observations were made. The silk I and a-helix forms are distinguishable by the chemical shifts as mentioned above. The silk II form may be distinguished from the silk I form by the 13C chemical shifts of the Ala Ca and Cb peaks, as well as the Gly Ca peak (Fig. 15) [10]. The 13C and 15N CP/MAS chemical shift data of silk ®broins are summarized in Table 2. A more quantitative analysis with the chemical shift will be described later. Fig. 16 shows the 13C CP/MAS spectrum of major ampullate silk of Nephila clavipes recorded in order to establish the dragline silk structure and to identify its crystalline regions [59]. The integrated areas of peaks from Gly, Ala, Gln and Tyr were 43, 46, 9 and 4% respectively, compared to values of 43, 30, 7 and 4% obtained by amino acid analysis of a small piece of the same sample. The detection of Ala, as well as the expected amounts of Gly, Gln and Tyr shows that both the crystalline and amorphous regions of silk

can be detected by 13C CP/MAS NMR. The chemical shifts of Ala in dragline silk demonstrate that Ala is present in a b-sheet. In fact, there is no evidence that any residues are present in the a-helix or random coil conformations. This observation is consistent with a model in which the amorphous regions of dragline silk may be present in very small (too small to diffract) sheet-like structures. The polymorphic structures of silk ®broin in the solid state were examined on the basis of a quantitative relationship between the 13C chemical shift and local structure in proteins. To determine this relationship, 13C chemical shift contour plots for Ca and Cb carbons of the Ala and Ser residues, and the Ca chemical shift plot for the Gly residues were prepared using atomic co-ordinates from the Protein Data Bank and 13 C NMR chemical shift data in aqueous solution reported for 40 proteins [28]. The construction of an extensive database of 13C Ca and Cb chemical shifts of proteins in solution (for proteins whose high-resolution crystal structures have

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Fig. 14. Partially-relaxed 50.3 MHz 13C solution NMR spectra of silk gland portion in intact B. mori mature larva at room temperature; t is delay time between the 1808 and 908 pulses [58].

Fig. 15. 50.3 MHz 13C CP/MAS NMR spectra of the Cp fraction of B. mori silk ®broin with silk I (A) and silk II forms (B) in the solid state [10].

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Table 2 15 N and 13C CP/MAS NMR chemical shifts (ppm) of the amino acid residues of B. mori silk ®broin with silk I and silk II forms, and those of S. c. ricini silk ®broin with a-helix and b-sheet forms [27] 15

13

N Chemical shifts

Silk I

a-helix

Silk II(b )

D

Gly

88.1

87.5

0.6

Ala

102.7

100.9 100.4

1.8 22.7

Ser

96.4

97.7

94.1

C Chemical shifts

a

2.3

Silk I Ca

43.8

CO Ca

170.7 51.4

Cb

16.5

CO

177.0

Ca Cb CO

58.0 60.7 173.7

a-helix 43.0 52.5 15.7 176.2

Silk II(b )

Da

43.1 42.8 169.5 49.4 48.6 20.2 20.0 172.3 171.8 55.4 63.6 (172.3) b

0.7 0.2 1.2 2.0 3.9 23.7 24.3 4.7 4.4 2.6 22.9 (1.4) b

a D(ppm) ˆ silk I (or a-helix)2silk II (or b-sheet) in chemical shifts. For example, a positive value means silk I peak is observed at lower ®eld than the silk II peak. b Superimposed on the Ala carbonyl peak.

been shown to be essentially the same as the solution structure) revealed results that are broadly similar to ab initio studies. The major factor affecting chemical shifts is the backbone geometry, which causes differences of

approximately 4 ppm between typical a-helix and bsheet geometries for Ca, and approximately 2 ppm for Cb. The side-chain torsion angle, x 1, has an effect of up to 0.5 ppm on the Ca shift, particularly for amino acids

Fig. 16. 50.3 MHz 13C CP/MAS NMR spectrum of major ampullate spider silk from N. clavipes in the solid state [59].

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317

Fig. 17. 50.3 MHz 13C CP/MAS NMR spectra of a-helix and b-sheet forms from S. c. ricini silk ®broin in the solid state. The Greek letters a and b stand for the peak position form a-helix and b-sheet, respectively [30].

Fig. 18. Contour plots of the conformation-dependent chemical shifts (ppm) of Ca and Cb carbons of Ala residues in 40 proteins. Chemical shift values in the region (21808 , f , 08, 21808 , c , 1808) are shown, where the density function is more than 1. Random coil chemical shifts are 50.0 ppm for Ala Ca carbon and 16.6 ppm for Ala Cb carbon [28].

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Fig. 19. Contour plots of the conformation-dependent chemical shifts (ppm) of Ca carbon of Gly residues in 40 proteins. Chemical shift values in the region …21808 , f , 1808; 21808 , c , 1808† are shown, where the density function is more than 1. Random coil chemical shifts are 42.7 ppm for Gly Ca carbon [28].

with branched side-chains at Cb. Hydrogen bonding to main-chain atoms has an effect of up to 0.9 ppm, which depends on the main-chain conformation. The different sequences of the protein and ring-current shifts from aromatic rings have an insigni®cant effect (except for residues following proline). There are signi®cant differences between different amino acid types in the backbone geometry dependence; the amino acids can be grouped together into ®ve different groups with different f and c shielding surfaces. The overall ®t of individual residues to a single nonresidue-speci®c surface, incorporating the effects of hydrogen bonding and x 1 angle, is 0.96 ppm for both Ca and Cb. The 13C CP/MAS NMR chemical shifts of the Ala, Ser and Gly residues of B. mori silk ®broin in silk I and silk II forms were used along with the 13C CP/ MAS NMR chemical shifts of the Ala residues of S. c. ricini silk ®broin in b-sheet and a-helix forms, for the structural analyses of silk ®broins (Figs. 15 and 17) [10,30]. Figs. 18 and 19 show the contour plots of the conformation-dependent chemical shifts (ppm) of Ca

and Cb carbons of Ala residue, and of Ca carbon of Gly residue in 40 proteins [28]. The random coil chemical shifts are 50 ppm for Ala Ca carbon, 16.6 ppm for Ala Cb carbon and 42.7 ppm for Gly Ca carbon. There is a clear conformational dependence of the chemical shifts. There are errors both in the experimental shifts determined from 13C CP/ MAS NMR experiments on silk ®broins and in the preparation process of the chemical shift contours. These errors have been combined, resulting in chemical shift error maps showing the root mean square difference between the observed Ca and Cb chemical shifts for the different silk ®broin structures and the estimated shifts as a function of f and c . The results for Ala residue are shown in Fig. 20 [28]. These angles are within the area surrounding by the 0.5 ppm line in the contour map for silk II. The allowed regions in the 13C chemical shift contour plots for Ca and Cb carbons of the Ala residue for the structures in silk ®broin with silk II, silk I and a-helix conformations, were determined using their 13 C isotropic NMR chemical shifts in the solid state.

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319

Fig. 20. Combined Ca and Cb chemical shifts error maps showing the root mean square difference between the observed Ala shifts of Silk I, ahelix and Silk II as a function of f and c . The area enclosed by a thick line in each plot is the area for which the combined error is less than 1ppm, and is thus the most likely conformation of Ala in Silk I, a-helix and Silk II, respectively [28].

There are two areas of the f and c map which satisfy the observed silk I chemical shift data for both the Ca and Cb carbons of the Ala residues in the 13C chemical shift contour plots [28,31]. 6. Determination of torsion angles for oriented silk ®ber by solid state NMR Many different experimental approaches have been used to characterize orientation in polymers, such as birefringence, ultraviolet±visible light spectroscopy, infrared dichroism, sonic modulus measurements, various X-ray techniques and nuclear magnetic resonance (NMR) [60]. Among these approaches, the rapid methodical developments of solid-state NMR methods have opened up new possibilities for such characterizations [60±66]. Orientation dependent NMR interactions such as chemical shielding, the dipole±dipole interaction, and the quadrupole interaction for nuclei with spin . 1/2, yield structural information on polymers in the solid-state. If these parameters are observed for each

site in oriented polymers, the solid-state NMR methods can give even the atomic coordinates of the polymers. This is especially effective in the determination of the atomic coordinates of peptides or proteins in membrane systems and ®brous proteins for which the X-ray technique gives only little structural information [67]. The solid-state NMR method has been applied to determine the torsion angles of peptide backbone of highly ordered silk ®broin ®ber and to obtain structural information of oriented polyamide ®bers. Asakura et al. [68] reported the structural analysis of uniaxially aligned polymers using solid-state 15N NMR method. Solid-state 15N NMR spectra were observed from blocks of oriented natural abundance silk ®broin, poly(p-phenylene terephthalamide) (PPTA), poly(g-benzyl-l-glutamate)(PBLG) and poly(m-phenylene isophthalamide) (PMIA) samples placed parallel to the applied magnetic ®eld. The u NH angles for silk ®broin, PPTA and PBLG are 908, 668 and 138. Fig. 21 gives the observed values of s parallel ± s iso for ®broin, PPTA and PBLG which are plotted versus u NH for 08 # u NH # 908: The shift to

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Fig. 21. Ranges of s parallel ± s iso vs u NH (0±908) searched over all a F and b F space. The regions painted by the horizontal lines are for natural abundance silk ®broin W, [ 15N]Gly-labeled silk ®broin X, and PBLG O and the region painted by the vertical lines is PPTA A. The u NH values and the observed s parallel ± s iso values for natural abundance silk ®broin , [ 15N]Gly-labeled silk ®broin , PPTA and PBLG are included. The three ranges for s parallel ± s iso determined from the spectrum of the block PMIA samples are represented by pairs of arrows [68].

higher ®eld by more than 120 ppm observed for silk ®broin compared with PBLG is a typical difference that would be expected between a b-sheet and ahelix, both with their long axes aligned parallel to the magnetic ®eld. The smaller difference in chemical shifts observed for silk ®broin and PPTA illustrates the sensitivity of s parallel to differences between similar secondary structures. The 13C± 15N dipolar splitting was observed for the oriented [1- 13C]Gly±[ 15N]Ala B. mori silk ®broin rods placed parallel to the magnetic ®eld with 15N solid-state NMR spectroscopy [25]. The isotope labeled and oriented silk ®broin samples were prepared by the cultivation of the middle silk gland from the silkworm. Fig. 22 shows 15N solid-state NMR spectrum (e) of the block [1- 13C]Gly± [ 15N]Ala silk ®broin rods whose macroscopic ®ber axes were placed parallel to B0 along with the spectra of [ 15N]Ala powder (d) and the block of single labeled [ 15N]Ala silk ®broin rods (b), whose ®ber axes were also placed parallel to B0. The asymmetric line shape of the spectrum (b) comes from the presence of a powder pattern (about 20%) and the distribution of ®ber axis [24]. The spectrum (c) is a subtraction of the spectrum (d) from the spectrum (e). In the spectrum (e), the peak at about 20 ppm is assigned

Fig. 22. Solid-state 40.4 MHz 15N NMR spectra of the block of [1- 13C]Gly±[ 15N]Ala silk ®broin rods (e) whose macroscopic ®ber axes were placed parallel to B0. The spectra of [ 15N]Ala powder (d) and the block of single labeled [ 15N]Ala silk ®broin rods (b), whose ®ber axes were also placed parallel to B0 are also shown. The spectra (a) and (c) are difference spectra, (c) ˆ (e)±(d) and (a) ˆ (c)±(b) [25].

to [ 15N]Ala which is involved in the silk ®broin rods. It is dif®cult to remove the [ 15N]Ala by only washing the silk gland after cultivation in water. By subtracting the spectrum (d) from the spectrum (e), the spectrum (c) without [ 15N]Ala peak is easily obtained. The spectrum (c) consists of a peak of [ 15N]Ala single labeled silk ®broin and two peaks due to [1- 13C]Gly±[ 15N]Ala dipolar splitting. Subtraction of the spectrum (b) from the spectrum (c), taking into account the 13C± 15N double labeling ratio allows the dipolar splitting to be determined as Dn obs ˆ 1:08 ^ 0:08 kHz (spectrum (a)). The observed splitting directly re¯ects the angle, Q N±C, between the 13 C± 15N peptide bonds and the macroscopic ®ber

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Fig. 23. Transformations from the principal axis system (PAS) to the molecular symmetry axis (MSA) system of (a) 15N and (b) 13C1 sites in a peptide plane; from the PAS to the ®ber axis system (FAS) of (c) 15N and (d) 13C1 sites; and from the FAS to the LAB frame of reference for 15N (e) and 13C (f) nuclei. The PAS is related to the FAS by the Euler angles a FX and b FX. This de®nition corresponds to setting gFX ˆ 08: Both 15N and 13C1 tensors have two tensor elements in the peptide plane as a result of the Euler angles a DNC and a DCN being 08. Consequently, s 22 and s 33 are the unique elements for the 15N and 13C1 tensors that do not lie in the peptide plane, respectively [26].

axis according to the following equation: Dn obs ˆ nuu …3cos2 Q N2C 2 1† where n is the dipolar coupling constant. The Q N±C angles determined here were 398, 1418, 768 and 1048. The error, ^0.08 kHz in Dn obs corresponds to ^28 error. Among these angles, 1418 is in agreement with the values, reported from X-ray diffraction analysis [12,18]. So although it is dif®cult to determine the structure of B. mori silk ®broin from only the dipolar coupling data, the number of unique orientations

321

possible for a given site can be reduced. By collecting the chemical shift tensor data of different sites for the oriented silk ®broins, it is possible to determine the torsion angles, f and c , for B. mori silk ®broin [69,70]. The chemical shift anisotropy (CSA) interaction for 15 N and 13C nuclei in a peptide plane can be interpreted with the chemical shift tensor transformation as shown in Fig. 23 [26,71]. The 15N and 13C CSA principal axis system (PAS) is the frame in which the CSA tensor is diagonal, with principal components s 11 , s 22 , s 33 : When the molecular symmetry axis (MSA) system is used as a reference frame, the Euler angles that express the relative orientations of the CSA, PAS and the MSA frames of reference are noted as a D and b D, where b DNC and b DCN notations are used for 15N and 13C nuclei, respectively as shown in Fig. 23(a) and (b). Inherent in this orientational relation is the assumption that s 33 lies in the peptide plane, yielding the result that g D ˆ 08 [72±75]. Here the ®ber axis system (FAS) is a reference frame ®xed in the aligned sample, and is de®ned such that the macroscopic ®ber axis lies in the Z direction (Fig. 23(c) and (d)). The CSA PAS is also at some orientation relative to the FAS, expressed by the Euler angles a F and b F. In this case, the location of X and Y within the plane perpendicular to the ®ber axis is de®ned such that s 33 lies in the XZ plane, where the Euler angles a F and b F for 13C and 15N nuclei are expressed by a FC and b FC and a FN and b FN as illustrated in Figs. 23(c) and (d). These positions also yield the convenient result that g FN ˆ gFC ˆ 08: The NMR spectra are observed in the laboratory frame of reference (LAB), in which the applied magnetic ®eld (B0) lies in the Z direction. The angles a L and b L are the Euler angles that transform the FAS into the LAB frame of reference (see Fig. 23(e) and (f)). Only two angles are required for this transformation because the NMR experiment is sensitive only to the component of the tensor parallel to B0. Therefore, the position of the FAS within the XY plane is arbitrary, and the third Euler angle, gL, can be conveniently set to zero. The angles a L and b L are set in the angular dependent NMR experiment performed here by placing the ®ber axis parallel …aL ˆ 08; bL ˆ 08† or perpendicular …08 , aL , 3608; bL ˆ 908† to B0. For the case where the ®ber axis is parallel to B0, aL ˆ 08 and bL ˆ 08; and the observed chemical shift

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spectral line shape. The spectra obtained from oriented protein ®ber samples aligned parallel and perpendicular to the magnetic ®eld yield eight possible orientations of the peptide plane relative to the long axis of the ®ber. Note that it is necessary to consider only one set of a F, b F angles per site in the spectral simulations since the remaining 7 possible pairs for each site will result in identical line shapes. A Gaussian probability distribution of ®ber axis orientations is employed to account for the spectral broadening observed in both the parallel and perpendicular cases. To obtain the bond orientation angles for N±H (u NH) and N±C 0 (u NC) of Gly and Ala residues relative to the ®ber axis (ZFAS), the following equation was used [24]: Fig. 24. Solid-state 40.4 MHz 15N NMR spectra of [ 15N]Ala B. mori silk ®broin aligned with the ®ber axis (a) parallel and (b) perpendicular to the magnetic ®eld, B0. The best-®t theoretical line shapes are superimposed on the experimental spectra. The distribution of ®ber axis orientations, p, was determined to be ^108 [26].

cosuNX ˆ cosbF cosbDNX

is given by

where a DNX and b DNX (X is H or C 0 ) are the Euler angles for the transformation from 15N PAS to molecular symmetry system (MSA) (Fig. 23(a)). The a DNC and b DNC for the [1- 13C]±[ 15N] double labeled samples are experimentally determined from solidstate 15N NMR observation. Similarly, to obtain the bond orientation angles for C 0 ˆ O (u CO) and C 0 ±N (u CN) of Gly and Ala residues relative to the ®ber axis, the following equation was used:

s par ˆ s 11 sin2 bF cos2 aF 1 s 22 sin2 bF sin2 aF 1 s 33 cos2 bF

…1†

For the case where the ®ber axis is perpendicular to B0, bL ˆ 908 and 08 , aL , 3608 (all values of a L will be equally represented in the spectrum). The observed chemical shift for this situation is given by [24] 2

s per …aF ; bF ; aL † ˆ F11 cos aL 1 2F12 cosaL sinaL 1 F22 sin2 aL

1 sinbF sinaF sinaDNX sinbDNX

…4†

cosuCX ˆ cosbF cosbDCX 1 sinbF cosaF cosaDCX sinbDCX

…2†

where F11 ˆ s 11 cos2 bF cos2 aF 1 s 22 cos2 bF sin2 aF 1 s 33 sin2 bF

1 sinbF cosaF cosaDNX sinbDNX

…3A†

F12 ˆ …s 22 2 s 11 †cosbF cosaF sinaF

…3B†

F22 ˆ s 11 sin2 aF 1 s 22 cos2 aF

…3C†

The Fij terms are i, j components of the CSA tensor expressed in the FAS reference frame. Eq. (2) represents a family of curves that are manifested in the

1 sinbF sinaF sinaDCX sinbDCX

…5†

where a DCX and b DCX (X is O or N) are the Euler angles from 13C PAS to MSA system Fig. 23(b). The a DCN and b DCN for the [ 15N]±[1- 13C] double labeled samples are experimentally determined from solid-state 13C NMR observation. The 15N labeled samples were obtained by the cultivation of the posterior silk glands in the amino acid medium containing [ 15N]Gly and [ 15N]Ala amino acids, respectively. By simulation, the 15N chemical shift tensor elements for the [ 15N]Gly and [ 15N]Ala residues were determined. Fig. 24 shows the 15N solid state NMR spectra of [ 15N]Ala silk ®broin ®ber

C. Zhao, T. Asakura / Progress in Nuclear Magnetic Resonance Spectroscopy 39 (2001) 301±352

Fig. 25. Solid-state 100.4 MHz 13C NMR spectra of an oriented block of [1- 13C]Gly B. mori ®broin ®bers as a function of the b L, the angle between the ®ber axis and B0. The best-®t theoretical line shapes obtained with the parameters, aFC ˆ 908; bFC ˆ 228 and p ˆ 118; are superimposed on the experimental spectra [26].

block aligned with the ®ber axis, which are set parallel (a) and perpendicular (b) to B0 [26]. The experimental spectra obtained from the oriented sample have had 20% of the spectral intensity subtracted as a powder pattern based on a 20% non-crystalline fraction in silk ®broin ®bers. By simulation of the spectra including the distribution of ®ber axis, p, the Euler angles between the 15N CSA principal axis and ®ber axis, a FN and b FN were determined. The 13C solid state NMR spectra of [1- 13C]Gly silk ®broin block ®bers are shown in Fig. 25 as a function of ®ber axis orientation with B0, BL. The structural parameters, a FC and b FC, are also obtained from the consistent simulation for the oriented block of [1- 13C]Ala silk ®broin. In order to determine the Euler angles of the 13C and 15N PAS relative to the

323

Fig. 26. Solid-state 100.4 MHz 13C (upper) and 40.4 MHz 15N (lower) powder pattern spectra of Boc±Gly±Ala±[1- 13C]Gly±[ 15N]Ala± Gly±Ala±OPac. The best-®t theoretical line shapes are superimposed on the experimental spectra. The a DCN and b DCN values for the [1- 13C]Gly were determined to be aDCN ˆ 08; bDCN ˆ 358: The 15N powder pattern spectrum revealed aDCN ˆ 08 and bDCN ˆ 1098 [26].

MSA system for [1- 13C]Gly, [ 15N]Ala sites, the (Boc± GA[1- 13C]G[ 15N]AGA±OPac) peptide was synthesized as a model compound for B. mori silk ®broin. Fig. 26 shows 13C (upper) and 15N (lower) powder pattern spectra of this model compound. The 13C powder pattern spectrum of [1- 13C]Gly is modi®ed by the dipolar interaction with [ 15N]Ala. The same dipolar interaction is observed in the 15N powder pattern spectrum. Euler angles of the 15N CSA PAS relative to the MSA system for the [1- 13C]Gly[ 15N]Ala double labeled model peptide can be determined from the simulation of 13C± 15N dipolar modulated powder pattern using the approach described by Teng et al. [71]. By the simulation for the 15N powder pattern spectrum including the dipolar interaction, the Euler angles of the 15N CSA PAS relative to the MSA system, a DNC and b DNC de®ned in Fig. 23(a) were determined as aDNC ˆ 08 ^ 58 and bDNC ˆ 1098 ^ 28: These angles indicate that the s 33 component of 15N nuclei is in the C±N±H

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Fig. 27. Variation in bond orientations, u NH, u NC, u CO and u CN as a function of torsion angles (f , c ) when the Ca(i21) ±Ca(i11) axis is constrained to be parallel to the FAS. The f ,c range shown is restricted to the generalized b-sheet region of the Ramachandran diagram [26].

plane. The Euler angles of the 13C CSA PAS relative to the MSA system for the [1- 13C]Gly[ 15N]Ala double labeled model peptide, a DCN and b DCN de®ned in Fig. 23(b) were determined as a DCN ˆ 908 ^ 58 and bDCN ˆ 358 ^ 28 from the simulation of the 13C powder pattern spectrum (Fig. 26, upper). These Euler angles indicate that the s 22 component of 13C nuclei is in the N±C±O plane. The Euler angles of both 15N and 13C CSA PAS relative to the MSA system of the [1- 13C]Ala[ 15N]Gly model compound (Boc±AG[1- 13C]A[ 15N]GAG± OPac) was also determined from the 15N and 13C powder pattern spectra, respectively. The Euler angles of the 13C and 15N CSA PAS relative to both the FAS and MSA system were determined experimentally for [1- 13C]Gly and [ 15N]Gly sites, and [1- 13C]Ala and [ 15N]Ala sites of the silk ®broin as mentioned above [76]. The bond orientations with respect to the ®ber axis, u NH and u NC, are calculated according to Eq. (4) with the Euler angles obtained here. For example, the u NH value is calculated with a FN, b FN, a DNH and b DNH. The u NH and u NC values of [ 15N]Gly and [ 15N]Ala sites were obtained in this way. In contrast, the bond orien-

tations, u CO and u CN, de®ned in Fig. 23 were calculated according to Eq. (5) with the Euler angles obtained here. The u CO and u CN values of [1- 13C]Gly and [1- 13C]Ala sites were then calculated. These yield eight possible orientations of the N±H bond, u NH, for the [ 15N]Gly labeled silk II structure relative to the ®ber axis. Previously a comparison of the u NH angle determined from the 15N chemical shift and 15N± 1H dipolar data has been necessary to eliminate four of the either a FN or b FN pairs. Two of the remaining four are simply related to one another by a 1808 rotation. Based on this data alone, it has not been possible to reduce the NMR-derived bond orientations to a unique solution [24]. Here, we reduce the possible solutions for the bond orientation angles of the Silk II structure by using the experimental observation from X-ray ®ber diffraction that the Ca(i 2 1) to Ca(i 1 1) axis is parallel to the ®ber axis [77]. This is based on the assumption of a two residue repeat unit along the peptide chain direction, in the ®ber diffraction unit cell. In this case, when we calculate the bond orientations of the ith residue, the orientations of two peptide planes with respect to the ®ber axis are necessary,

C. Zhao, T. Asakura / Progress in Nuclear Magnetic Resonance Spectroscopy 39 (2001) 301±352 13

Fig. 28. Variation in bond orientations constrained by the Ca(i21) ± Ca(i11) axis being parallel to the FAS and by the NMR orientational constraints for the (a) Ala and (b) Gly residues. The conformational space is restricted to an experimental error of ^58 for each bond orientation [26].

namely, the orientation of the ®rst plane, Ca(i 2 1)± C 0 (i 2 1)±N(i)±Ca(i) and the second plane, Ca(i)± C 0 (i)±N(i 1 1)±Ca(i 1 1) with respect to the Ca(i 1 1)±Ca(i 2 1) axis can be calculated with the atomic coordinates that are generated as a function of torsion angles (f ,c ). Thus, the torsion angle of the Gly residue can be de®ned using the repeat unit, Ala Ca(i 2 1)±Gly(i)±AlaCa(i 1 1), and the torsion angle of the Ala residue can be de®ned using the repeat unit, GlyCa(i 2 1)±Ala(i)±GlyCa(i 1 1). Fig. 27 shows the variation of bond orientations, u NH, u NC, u CO and u CN, with respect to the Ca(i 2 1)±Ca(i 1 1) direction, as a function of torsion angles (f ,c ). The maximum and minimum ranges of u values within 21808 , f , 08 and 08 , c , 1808, corresponding to the b-sheet structural region, are calculated as 24.58 , u NH , 114.38, 5.78 , u NC , 95.88, 68.78 , u CO , 152.58, and 84.58 , u CN , 168.38. For these ranges, unique combinations of u NH and u NC, and u CO and u CN are de®ned. Only one combination for each of u NH and u NC are de®ned (u NH ˆ 888; uNC ˆ 408 for [ 15N]Gly site and uNH ˆ 838; uNC ˆ 398 for [ 15N]Ala site). Similarly, one combination of u CO and u CN is

325

de®ned for [1- C]Gly site …uCO ˆ 898; uCN ˆ 1398†: In the case of [1- 13C]Ala site, there are two possibilities for the u CO and u CN values, uCO ˆ 898; uCN ˆ 1428 or uCO ˆ 928; uCN ˆ 1458: However, these two possibilities are equal within experimental error (^58). The 13C± 15N bond orientation of uniaxially oriented B. mori silk ®broin has been reported [25] based on the 13C± 15N dipolar splitting of [1- 13C]Gly[ 15N]Ala double labeled sample using solid state 15N NMR. This data de®ned two possible orientations of 398 and 1418 for the u NC of Ala residue. This result is in agreement with the reduced value for [ 15N]Ala u NC (398) calculated, indicating accuracy of the reducing process and these results. In addition, since the ([1- 13C]Gly[ 15N]Ala) sequence repeats in the silk ®broin, the u CN value for the Gly residue can be recalculated to be 1418 with the u NC value (398) for Ala residue as uCN ˆ 1808 2 uNC : This value of 1418 also agrees with the de®ned data, 1398 for Gly residue. The speci®c bond orientations, u NH, u NC, u CO and u CN, are easily calculated as a function of torsion angles, f and c as mentioned above. Especially, the orientation of ®rst peptide plane of the ith residue, Ca(i 2 1)±C 0 (i 2 1)±N(i)±Ca(i), is de®ned with two bond orientations, N(i)±H and N(i)±C 0 (i 2 1), i.e., u NH and u NC, and the second peptide plane, Ca(i)± C 0 (i)±N(i 1 1)±Ca(i 1 1), is de®ned with the bond orientations, C 0 (i)±O and C 0 (i)±N(i 1 1), i.e., u CO and u CN. Thus, from the comparison of these two peptide plane orientations within the Ca(i 2 1)± Ca(i 1 1) which corresponds to a ®ber repeat unit cell with the observed data, we can pick up the constrained f ± c pairs (see below). Fig. 28(a) shows the calculated region of four kinds of u values for Ala site which is satis®ed with experimental data (u NH ˆ 83 ^ 58 and uNC ˆ 39 ^ 58 of [ 15N]Ala site, and uCO ˆ 89 ^ 58 and uCN ˆ 142 ^ 58 of [ 15N]Ala site). In this calculation, the Gly Ca(i 2 1)±Gly Ca(i 1 1) direction was set parallel to the ®ber axis based on the X-ray diffraction data. The width of each dotted line corresponds to experimental error within ^58 for each u value. There is only one region (A) under the b-sheet condition 1808 , f , 08 and 08 , c , 1808) and the best ®t torsion angles (f ,c ) of Ala residue in this constrained region A are obtained as (21408,1428) based on minimum error of u values. The Gly Ca(i 2 1)±Gly Ca(i 1 1)

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Table 3 Torsion angles (f , c ) of silk II structure of B. mori silk ®broin determined from solid-state NMR and other methods [26] Method

f (deg)

c (deg)

Ê) Ca(i21) ±Ca(i11) distance (A

Sum of u err a (deg)

NMR and ®ber diffraction This work Ala Gly

2140 2139

142 135

6.98 6.92

4.0 4.8

2139 2139

140 140

2134 2150

154 142

2149 2150

148 146

X-ray ®ber diffraction Marsh's model b Ala Gly Takahashi's model d Ala Gly Energy calculation Fossey's model e Ala Gly a b c d e

6.94 c 6.98 c

7.06

Sum of u err is the sum of four minimum errors between observed and calculated m values u NH, u NC, u CO and u CN at the best ®t torsion angle. Ref. [12]. Unit cell length. Ref. [18]. Ref. [21].

Fig. 29. Solid-state 40.4 MHz 15N NMR spectra of an oriented block of [ 15N]Gly S. c. ricini silk ®broin ®ber (A) as a function of the angle between the ®ber axis and the magnetic ®eld B0. Non-crystalline fraction in the silk ®broin ®bers has been determined by subtracting the powder pattern from these spectra. The simulated spectra are shown in (B). The distribution of the ®ber axis orientation, p, was determined to be 318 [79].

distance, which corresponds to unit cell length along Ê with the C axis (®ber axis) is calculated to be 6.98 A best ®tted torsion angles (f ,c ) of Ala determined here, indicating fairly good agreement with X-ray ®ber diffraction data [12,18,77,78]. Similar analysis was performed for the Gly site of silk ®broin with silk II structure as shown in Fig. 28(b), where the bond orientation angles u for Gly site used here were u NH ˆ 88 ^ 58; uNC ˆ 40 ^ 58; uCO ˆ 89 ^ 58 and uCN ˆ 139 ^ 58: In this case, Ala Ca(i 2 1)Ala Ca(i 1 1) direction is set parallel to the ®ber axis. The f ± c dependency of each u values of Gly is similar to that of the Ala site (Fig. 28(a)). The best ®t torsion angle (f ,c ) of Gly residue in the constrained region A is obtained as (21398, 1358). The Ala Ca(i 2 1)±Ala Ca(i 1 1) distance Ê with the best ®t torsion was calculated to be 6.91 A angle (f ,c ) of Gly residue. The ®nal results are listed in Table 3 [26]. The structure of silk ®broin ®ber from a wild silkworm, S. c. ricini, in which the amino acid sequence is similar to the spider (major ampullate) silk, was also determined by Asakura et al. with solid-state NMR [79]. The 15N and 13C labeled samples for the dominant

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Fig. 30. Solid-state 100.0 MHz 13C NMR spectra of an oriented block of (A) [1- 13C]Gly S. c. ricini silk ®broin ®ber as a function of the angle between the ®ber axis and the magnetic ®eld B0. The non-crystalline fraction in the silk ®broin ®bers has been determined by subtracting the powder pattern from these spectra. The simulated spectra are shown in (B). The distribution of ®ber axis orientation, p, was determined to be 318 [79].

amino acids, Ala and Gly, residues of S. c. ricini silk ®bers were obtained by oral administration of [ 15N]Ala, [1- 13C]Ala, [ 15N]Gly, or [1- 13C]Gly to the ®fth instar larvae of the silkworm. The blocks of the oriented silk ®bers stretched by about 10 times the original length of the sample, were prepared and the 15 N and 13C solid-state NMR were observed by changing the angles of the oriented silk ®ber axis and the magnetic ®eld. Fig. 29 is a series of spectra Gly of oriented components of [ 15N] silk ®broin ®ber block obtained by subtraction of the powder pattern components from the oriented spectra, along with the simulated spectra used for determination of the Euler angles, a F and b F. The fraction of oriented components is 75% for Ala site and 65% for the Gly site. The p values are larger for [ 15N]Gly silk ®broin ®ber than for [ 15N]Ala silk ®broin ®ber. The a F and b F values are almost the same for the two 15N sites and are almost the same as those for B. mori silk ®broin ®ber. Fig. 30 shows a series of spectra of oriented components of [1- 13C]Gly silk ®broin ®ber block obtained by subtraction of the powder pattern components from the original oriented spectra in Fig. 29

Fig. 31. Variation in bond orientations, u NH, u NC, u CO and u CN as a function of torsion angles (f , c ) constrained by the Ca(i21) ±Ca(i11) axis being parallel to the FAS and by NMR orientational constraints for (A)Ala and (B)Gly residues. The conformational space is restricted to an experimental error of ^58 for each bond orientation [79].

along with the simulated spectra for determination of the Euler angles, a F and b F. The fraction of the powder components was determined to be 35%, which is in agreement with the 15N Gly data. The p value for [1- 13C] Ala silk ®broin ®ber block is 23 and is smaller than the value, 31, for [1- 13C]Gly silk ®broin ®ber block. A p value of 10 was reported for oriented B. mori silk ®broin ®bers, and therefore the distribution of the ®ber axis is larger for the S. c. ricini silk ®ber than for the B. mori silk ®ber even in the Ala

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Fig. 32. Angle g between the Cb ± 2H bond vector and the ®ber axis calculated as a function of the torsion angle x around the N±Ca ±Cb ±O axis, based on a uniaxially oriented b-sheet protein structure [80].

sites. Fig. 31 gives the determination of f ± c angles for Ala and Gly residues of S. c. ricini silk ®broin, taking into account the experimental error. Deuterium solid-state NMR was used to study the dynamics and molecular structure of the serine side chains in silk ®broin from B. mori and from S. c. ricini. Quadrupole echo 2H NMR powder spectra were simulated as a function of the exchange rate and libration angle, according to an appropriate

dynamic model. The electric ®eld gradient tensor of a deuterium substituent is virtually axially symmetric and aligned along the C- 2H bond axis. Therefore, the angle u of this C- 2H bond vector with respect to the magnetic ®eld can be determined directly from the spectral quadrupole splitting Dn Q, according to Dn Q ˆ 3=4 Qcc …3 cos2 u 2 1†; where Qcc is the rigid lattice quadrupole coupling constant. Similarly, in the case of a ®ber that is oriented parallel to the magnetic ®eld, all C± 2H bond vectors lie along the rim of a cone around the ®ber axis with an angle g that is equivalent to u . Fig. 32 illustrates the relationship between x and g assuming standard peptide bond lengths and angles [80]. The angle was calculated by rotating the torsion angle x for the Ca ±Cb axis over 3608 in 0.58 increments. The two lines in the graph correspond to the two Ca ± 2H substituents on Ser, which differ only in terms of a phase shift along x . When the ®ber axis is aligned parallel …a ˆ 08†; u is equal to g and can be determined directly from the observed quadrupole splitting. Using this correlation, the expected line shape of the titled sample …a ˆ 908† has then calculated and the simulated spectra were used to ®t and con®rm the experimental date. Fig. 33 shows the 2H NMR spectra of uniaxially aligned [3,3- 2H2]Ser labeled B. mori silk ®broin, with the ®ber axis set parallel …a ˆ 08† and perpendicular …a ˆ 908† to the magnetic ®eld direction, respectively [80]. The line shapes show an appreciable dependence on the angle a , which indicates that Ser side chains are indeed reasonably well oriented with respect to the ®ber axis. Uniaxially oriented silk ®bers were used to determine the side-chain conformation and the orientational distribution of the Ser residues in the slow motional component of B. mori silk ®broin. The gauche 1 conformer around N±Ca ±Cb ±O was found to be dominant, suggesting that the hydroxyl groups of Ser interact with carbonyl groups on adjacent chains and thereby contribute to the intermolecular hydrogen-bonding network of the ®ber. 7. Determination of torsion angles for silk by spindiffusion NMR

Fig. 33. Solid-state 61.06 MHz 2H NMR quadrupole echo spectra of uniaxially aligned [3,3- 2H2]Ser-labeled B. mori silk ®broin. The ®ber axis was set parallel (a) and perpendicular (b) to the magnetic ®eld direction [80].

Solid-state NMR can probe the local structure in disordered systems where the lack of translational long-range order makes the application of a diffraction

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Fig. 34. Simulations of 2D spin-diffusion powder spectra at quasi-equilibrium for the 13CyO resonance in Gly. The following structures were assumed: (a) amorphous spectrum; (b) isolated a-helix (or fully ordered array thereof); (c) 31-helix using Euler angles between the CSA principal axis systems of a ˆ 1148; b ˆ 788 and g ˆ 3468; (d) uniaxially ordered array of 21-helices, taking into account 50 nuclei and assuming that the helix axis coincides with the principal axis belonging the s 11; (e) uniaxially ordered array of a-helices, taking into account 50 nuclei and assuming that the helix is parallel to s 22; (f) uniaxially ordered array of 31-helices, taking into account 50 nuclei and the Euler angles. The simulations were performed in the frequency domain using the simulation program 'gamma'. For the simulations the same resolution (1.76 points /ppm) as in the experimental spectra was chosen [100].

technique dif®cult [81]. The most useful NMR interaction for structural studies is the magnetic dipole interaction because of its simple and quantitative relationship with the molecular structure of the material at the atomic level. In the simplest case, for an isolated spin pair, the dipolar interaction manifests itself as a line splitting from which the internuclear distance can directly be evaluated. If the spectral resolution is insuf®cient, the investigation of the dynamics of the dipolar-induced polarization transfer between the

nuclei can be used to determine the distance [82± 84]. For samples with a regular or random distribution of many coupled spins the spatial transfer of polarization, often called spin-diffusion [85], is the most useful source of geometrical information on the local structure [64,84,86±94]. Combined with twodimensional (2D) NMR spectroscopy [95,96], it can be used to determine the relative orientation of neighbouring molecular segments in crystalline and amorphous materials [89±94,97,98]. The resulting

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Fig. 35. (a) Experimental 100.6 MHz 13C spectrum of [1- 13C]Gly, re-plotted for direct comparison with (b)±(e). (b) Best-®t of the experimental spectrum by a two-site exchange model with an amorphous background. The conformation represents, within reasonable expectations, a 31helical structure. (c) Difference between the experimental spectrum (a) and ®t (b). (d) Best-®t by a super-position of an amorphous and a diagonal spectrum. (e) Difference between (a) and (d) [100].

2D spin-diffusion correlation maps directly relate to the local geometry if the purely spectral contributions are corrected for, or eliminated by, suitable pulse schemes [84,94,99]. The local structure of dragline silk from the spider Nephila madagascariensis has been investigated by solid-state NMR [100]. Fig. 34 shows a simulation of 2D spin-diffusion powder spectra at quasi-equilibrium for the 13CyO resonance in Gly. The following structures were assumed: (a) amorphous spectrum; (b)

isolated a-helix (or fully ordered array thereof); (c) 31-helix using Euler angles between the CSA principal axis systems of a ˆ 1148; b ˆ 788; and g ˆ 3468; (d) uniaxially ordered array of 21-helices, taking into account 50 nuclei and assuming that the helix axis coincides with the principal axis belonging to s 11; (e) uniaxially ordered array of a-helices, taking into account 50 nuclei and assuming that the helix is parallel to s 22; (f) uniaxially ordered array of 31-helices, taking into account 50 nuclei and using the Euler

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Fig. 36. Experimental 100.0 MHz 13C DOQSY spectra of S. c. ricini silk ®broin. a, b, Spectra for ®ber (a) and ®lm (b) samples. c, d, Best ®t for the ®ber (c) and ®lm (d) samples. These include the chemical shift bias but are identical to the ones without bias at the resolution of the contour plot. e, f, Differences between best ®t and experimental data for ®ber (e) and ®lm (f). For spectra a±d, the total signal intensity of the carbonyl region in the 2D spectra in normalized to 1000 for a resolution of 1.76 ppm per point. The levels are absolute levels and are set at 0.025, 0.050, 0.075 and so on, to enable direct comparison. The principal values of the 13CyO CSA tensors used in the ®t were obtained from static 1D powder patterns and amounted to (224.8, 194.7, 91.6 ppm) for ®lm and (244.8, 184.5, 89.2 ppm) for the ®ber sample [36].

angles. The simulations were performed in the frequency domain using the simulation program `gamma' and following the general scheme of the example given in Ref. [101]. For the simulations the same resolution (1.76 points/ppm) as in the experimental spectra was chosen. Two-dimensional (2D) spin-diffusion experiments show that the alaninerich domains of the protein from b-sheet structures are in agreement with one-dimensional NMR results from a different species of the genus Nephila, but at variance with diffraction results. For an a-helical structure as seen in Fig. 35(a), all CyO bond direc-

tions are approximately parallel to the helical chain axis. Correspondingly, the principal axis directions associated with the Cartrsian s yy components of all 13 C CSA tensors coincide approximately with the helix axis. The relative tensor orientation between two neighbouring carboxylic CSA tensors is therefore described by a rotation around the y axis of the principal axis system of the CSA tensor, corresponding to a set of Euler angles (08, 1008, 08). From a simulated poly(G±G±X) a-helix (with X ˆ Ala), obtained with the program `quanta' [102], Euler angles of (65.48, 97.78, 189.98) ˆ (214.68, 97.78, 9.98) are extracted.

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Fig. 37. REDOR dephasing curves for natural abundance carbon peaks in Silk B (Table 4). Experimental data show along with best-®t incorporating nitrogen neighbors at two distances [110].

The quasi-equilibrium spectrum of an isolated (G±G± X)n a-helical structure, where 3.6 residues constitute one turn and the helix is repeated after ®ve turns, is described by a polarization exchange between 12 Gly sites (the X residues are not 13C labeled). Fig. 34(b) shows the simulated quasi-equilibrium spectra for spin-diffusion between all 13C in an isolated helix. For the 31-helixal arrangement as seen in Fig. 35(b), such a structure is found, for example, for the form II crystal structures of poly±Gly, poly(L±A±G±G), poly(L±A±G±G±G), and poly(L±G± b ±A). In a (G±G±X)m-31-helical structure only two symmetrically inequivalent [1- 13C]Gly sites exist and the quasi-equilibrium spectrum corresponds to a two site exchange spectrum. From model (G±G±A)n poly-

peptides, the Euler angles that relate the OyC±N fragments of each Gly residue were found to be a ˆ 1148; b ˆ 788 and g ˆ 3468: A simulated quasi-equilibrium 2D spin-diffusion spectrum assuming spin exchange between Gly sites related by these Euler angles is depicted in Fig. 34(c). Fig. 35(a) shows the experimental spectrum of [1- 13C]Gly. The best ®t, shown in Fig. 35(b), reproduces the off-diagonal features of the experimental 2D spin-diffusion spectrum remarkably well. The ®tted Euler angles are a ˆ 101 ^ 138; b ˆ 62 ^ 148 and g ˆ 333 ^ 108 (error estimated at 90% con®dential limit) and an admixture of 14 ^ 5% signal intensity from an isotopic exchange pattern. The difference spectrum is depicted in Fig. 35(c) and gives no indication that the quasi-equilibrium

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Table 4 Distances and fractions of peak intensity from Ala and Gly peaks [110] Ê) Distance (A

Fraction

Silk A

Ê) Distance (A

Fraction

Silk B

Alanine Cb 2:6 ^ 0:2 4:0 ^ 0:3

0:10 ^ 0:04 0:60 ^ 0:1

Alanine Cb 2:39 ^ 0:1 3:77 ^ 0:1

0:10 ^ 0:02 0:39 ^ 0:05

Glycine Ca 1:51 ^ 0:05 3:5 ^ 0:3

0:31 ^ 0:02 0:66 ^ 0:2

Glycine Ca 1:47 ^ 0:2 3:9 ^ 0:3

0:12 ^ 0:01 0:7 ^ 0:3

spectrum is distorted through the in¯uence of the factor F ij …0† which would lead to a monotonic decrease of the cross-peak intensity with increasing distance from the main diagonal of the spectrum. Furthermore, the secondary structure of S. c. ricini silk has been determined by solid-state NMR [36]. Beek et al. adapt a recently developed solid-state NMR technique to determine torsion angle pair (f ,q ) in the protein backbone, and study the distribution of conformations in S. c. ricini silk. Although the most probable conformation in native ®bers is an antiparallel b-sheet, ®lm produced from liquid directly extracted from the silk glands appears to be primarily a-helix. The experimental DOQSY spectra [103] for both ®ber (Fig. 36(a)) and ®lm (Fig. 36(b)) samples and the best ®ts are shown in Fig. 36(c) and (d), respectively. The difference spectra are shown in Fig. 36(e) and (f). The most probable conformation is …f; c† ˆ …21358; 1508†; and 70% of P…f; c† fall within the b-sheet region. The intensity around (21808, 1808) is attributed to inter-strand contacts

within the b-sheet. For the ®lm, it shows a maximum at (^608, ^458) corresponding to 60% of P…f; c†. Within the 15 grid resolution of their analysis, these regions of the Ramachandran plot coincide with the standard conformation for the anti-parallel b-sheet (21408, 1358) and for the a-helix (2578, 2478).

8. Determination of the atomic distance of silk model peptides using REDOR One of the most powerful capabilities of solid-state NMR is the measurement of internuclear distances from dipolar coupling in the solid state. The advance of routine magic-angle spinning to enhance sensitivity and average chemical shift anisotropies has made it possible to obtain inter-atomic distances by observing the nuclear Overhauser enhancements (NOEs) between hydrogen atoms in the solid-state. Alternatively, the detection of weak hetero-nuclear dipole interactions, such as between 13C and 15N nuclei,

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Fig. 38. 100.3 MHz 13C CP/MAS spectrum of (AG)15 with silk I form after dialysis of the 9 M LiBr solution against water. The symbol `ss' means spinning side band [31].

may be used to obtain distance information: an analogous technique known as rotational±echo double resonance (REDOR) was developed from measuring hetero-nuclear dipolar couplings in rotating solids [104±106]. Similarly, the observation of dipole interactions between like nuclei may be used. When samples can be prepared with isolated pairs of isotope spin labels, the method gives quantitative distance measurements. The distance information r derives from the dipolar coupling, which varies as 1=r 3 : Several examples now exist in the biopolymer literature, including Graminicidin A [107], Saccharomyces cerevisiae tridecapeptide mating pheromone [108] and EPSP synthase [109]. In general, using 13C and 15 Ê can be determined N labels, distances out to ca. 5 A Ê to an accuracy of 0.1 A, with even better accuracy for shorter distance.

Jelinski et al. used the REDOR NMR on the strategically 13C and 15N labeled samples to study the conformation of the LGXQ (X ˆ S, G, or N) motif in the major ampullate gland dragline silk form the spider N. clavipes [110]. Fig. 37 shows the dephasing curves constructed from Ala Cb and Gly Ca in Silk B sample. The solid lines are best ®ts incorporating two distances and associated fractions of the peak intensity. The simulations are actually superpositions of three curves, where the third curve represents 13C nuclei that have both 15N neighbours present. The parameters found from the ®tting routines are given in Table 4. In each case, the shorter distance is in excellent agreement with the distance Ê for Cb expected to the residue's own nitrogen (2.45 A Ê for Ca), and the associated fractions of and 1.45 A peak intensity give 15N labelling estimates for these found from the solution NMR and suggest that the Ala residues are ,10% labeled in each sample, while the Gly residues are 30% labeled in Silk A and 12% labeled in Silk B. A method is described for calculating REDOR dephasing curves suitable for background subtractions, using probability distributions of nitrogen atoms surrounding a given carbon site, which are developed from coordinates in the Brookhaven Protein Data Bank. The validity of the method is established by comparison with the dephasing observed on natural abundance 13C peaks for Gly and Ala. Straightforward ®tting of universal REDOR dephasing curves to the background corrected peaks of interest provide results which are not self-consistent, and a more sophisticated analysis shows that there is probably some heterogeneity in the structures formed by the LGXQ sequences, the data indicate that they all form compact turn-like structures.

Table 5 Several 13C and 15N isotope labeled Ala and Gly alternating copolypeptides, (AG)15 synthesized by solid phase methods for 2D spin-diffusion and REDOR experiments [31] Peptide (AG)6 (AG)7 (AG)6 (AG)6 (AG)6 (AG)7 (AG)6

13

13

A[1- C]G14[1- C]A15 G(AG)7 [1- 13C]A15[1- 13C]G16 (AG)7 [1- 13C]A13[1- 13C] G14 (AG)8 A[1- 13C]G14A15[1- 13C]G16(AG)7 A[1- 13C]G14A[ 15N]G16 (AG)7 [1- 13C]A15G[ 15N]A17 G(AG)6 A[1- 13C]G14AG[ 15N]A17 G(AG)6

Method

Information

Spin±diffusion Spin±duffusion Spin±diffusion Spin±diffusion REDOR REDOR REDOR

Ala15 (f ,c ) Gly16 (f ,c ) Gly14 (f ,c ) Ala15 (f ,c ), Gly16 (f ,c ) [1- 13C]G14´´ ´[ 15N]G16 [1- 13C]A15´´ ´[ 15N]A17 [1- 13C]G14´´ ´[ 15N]A17

Angle Angle Angle Angle Distance Distance Distance

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Fig. 39. The experimental (a) and simulated (b) 2D spin-diffusion 100.0 MHz 13C NMR spectra of (AG)6A[1- 13C]G14[1- 13C]A15G(AG)7. The mixing time of 2 s was optimized for the experiment. The torsion angles (f and c ) used for the simulation of this spectrum were …f; c† ˆ …2608; 1308† for the Ala residue.

In the past few years, the REDOR techniques have been developed to determine internuclear distances in biological solids such as peptides, proteins aggregates and membrane proteins [111±119]. The REDOR technique was used to determine the torsion angles describing the backbone conformation of a hexapeptide of the sequence, AGSGAG, with the wellcharacterised silk II conformation [120]. 13C and 15 N-isotopic labelling was performed such that the measured 13C± 15N distance will place limits on the possible values of the f and c angles of the Ala residue at the 5th position. The torsion angles of the Ala residue were reported as f ˆ 21428 and c ˆ 1458: Using these torsion angles, the distance between the 13C and 15N labels in the selectively labelled hexaÊ . The results of the REDOR experipeptide is 4.55 A Ê, ments gave an intra-molecular distance of 4.32 A which is in good agreement with the predicted value for the silk II conformation.

Fig. 40. The calculated 2D spin-diffusion NMR spectra of Ala residues in two areas selected from the previous chemical shift analysis. The calculation of the spectra was performed for each 208 [28,31].

9. Determination of Silk I (B. mori silk structure before spinning in the solid state) by combination of several solid state NMR methods Despite a long history of interest in the less stable

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Fig. 41. The experimental 2D spin-diffusion 100.3 MHz 13C NMR spectra of (AG)6 [1- 13C]A13[1- 13C]G14(AG)8 (a) and (AG)7[1- 13C]A15[1- 13C]G16(AG)7 (b). The mixing time of 2 s. was optimized for the experiment. The calculated spectrum (c) of the Gly residue with torsion angles …f; c† ˆ …708; 308† is also shown [31].

silk I form, its structure remains poorly understood [8,17,19±21,29,58,121,122], because attempts to induce orientation of the silk I form for studies by X-ray and electron diffraction cause the silk I form to convert to the more stable silk II form easily. Consequently, structural investigations of the silk I form have been based on model building of peptides

such as (AG)n and comparison with limited experimental data, resulting in a number of con¯icting models describing the structure of silk I [17,19± 21,29,121]. The wide-angle X-ray scattering (WAXS) pattern calculated for these models could not reproduce the same intensity pattern as observed for silk I [122], and some of the models are in con¯ict

Fig. 42. The Ramachandran map of the calculated 2D spin-diffusion NMR spectra as a function of (f ,c ) of Gly residue for each 208. There are four (f ,c ) regions, that is, (2120 to 21008, 40±608), (280 to 2608, 240 to 2208), (60±808, 20±408) and (100±1208, 260 to 2408) which give agreement between the calculated and observed 2D spin-diffusion spectra. The area with enough data points to give reliable chemical shift predictions for the Ca carbon chemical shifts of Gly residue is also shown [28,31].

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Fig. 43. The observed 2D spin-diffusion 100.0 MHz 13C spectrum of (AG)6 A[1- 13C]G14A[1- 13C]G16(AG)7 (left upper) and calculated spectrum (right upper) of the Gly16 residue with torsion angles …f; c† ˆ …708; 308† is also shown when the torsion angles are …f; c† ˆ …2608; 1308† for the Ala15 residue. The calculated spectra are also shown as a function of f and c angles of the Gly residue for each 108 for four regions selected in Fig. 42 [31].

with the 13C chemical shift data, used to predict the backbone conformation [27,28,31]. Here, we have combined the results from several solid state NMR techniques to determine the conformation of un-oriented samples of a model peptide (AG)15 in the silk I form: we used solid-state two dimensional spindiffusion NMR under off-magic angle spinning condi-

tion and REDOR [31], together with the NMR chemical shift-structure relationships reported previously. The conformation-dependent 13C NMR chemical shifts, especially of the Ala and Ser residues of silk ®broins and related model compounds, have been successfully used to determine the conformation of several types of silks.

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Fig. 44. Two experimental 13C REDOR difference curves DS/S0 for samples (AG)6 A[1- 13C]G14A[ 15N]G16(AG)7; K, and (AG)7[1- 13C]A15G[ 15N]A17G (AG)6; O. Solid lines show theoretical dephasing curves corresponding to the designated distances. By comparing the REDOR data and the theoretical dephasing curves, the 13C± 15N inter-atomic distances for the samples (AG)6A[1- 13C]G14A[ 15N]G16(AG)7 and (AG)7[1- 13C]A15G[ 15N]A17G(AG)6 were Ê , respectively [31]. determined to be 3.2 ^ 0.1 and 3.8 ^ 0.1 A

Fig. 38 shows the 13C CP/MAS spectrum of (AG)15 after dissolving the peptide in 9 M LiBr and then dialyzing against water. The sharp peaks with the chemical shifts, 16.7 ppm for Cb, 50.8 ppm for Ca and 176.8 ppm for carbonyl carbons of Ala residue, respectively, and 43.4 ppm for Ca and 169.9 ppm for carbonyl carbons of Gly residue, respectively, observed in the spectrum indicate clearly that the structure of the sample, (AG)15, is exclusively silk I [8,17,28,29]. The chemical shift data shown here are different from those in the original recent papers [11,31] because of the changing chemical shift reference for the chemical shift contour plots (Figs. 18 and 19) [28]. The IR spectrum is also the same as the pattern of silk I reported previously although it is not shown here [17]. All of the other isotope-labeled 30 mers used here and shown in Table 5 also take silk I structure. The 2D spin-diffusion NMR spectrum (only the carbonyl region was expanded) of (AG)6A[1- 13C] G14[1- 13C]A15G(AG)7(a) is shown in Fig. 39 together with the simulated spectrum for the torsion angles (f and c ˆ 2608 and 1308) of Ala residue (b). For the calculation of the 2D spin-diffusion spectra, it is necessary to obtain the principal values of the chemical shift tensors of the carbonyl carbon. Thus, by observing the sample under slow MAS, the principal values of the chemical shift for Ala, and Gly carbonyl carbons were

determined [31,123]. The 2D spin-diffusion spectra were calculated as a function of f and c of the Ala residues of (AG)15 for each 20 degree in two areas in the Ramachandran map shown in Fig. 40 [28,31]. These two areas were selected to satisfy both Ca and Cb chemical shifts of Ala residue of the silk ®broin with silk I form; namely, the root mean square difference between the observed Ca and Cb chemical shifts for the different silk ®broin structures and the estimated shifts from the chemical shift contour maps of Ala residues in proteins was calculated and the area indicated by the lines with a combined chemical shift error of less than 1 ppm was selected (Fig. 20). The calculated spectra in the area (f ˆ 280 to 2208, c ˆ 90 to 1808) clearly resemble the experimental spectra. The detailed simulation was also performed for each 108 in this area. By ®tting the calculated spectra to the experimental one, the torsion angles, (2608, 1308) for Ala residue were determined as shown in Fig. 39. Thus, the experimental error in the determination of the angles is ^58. Fig. 41 shows the experimental 2D spin-diffusion NMR spectra of (AG)6 [1- 13C]A13[1- 13C]G14(AG)8 (a) and (AG)7[1- 13C]A15[1- 13C]G16(AG)7 (b) together with the simulated spectrum for the torsion angles …f ˆ 708; c ˆ 308† of the Gly residue(c). The observed spectra are essentially the same, indicating that all of the torsion angles of the Gly residues in (AG)15 are expected to be the same. The calculated spectra are shown as a function of (f ,c ) of Gly residues for each 20 degree interval in the Ramachandran map (Fig. 42). The area with enough data points to give reliable chemical shift predictions for the Ca carbon chemical shifts of Gly residue, in which the density function is greater than 1, is also shown [28]. However, contrary to the case of Ca and Cb carbons of Ala residue, the conformation-dependence of the Gly Ca chemical shift is small and therefore the chemical shift maps are not used for selecting the area of silk I structure in the Ramachandran map. There are four candidate areas in which the calculated spectra most closely resemble the experimental spin-diffusion spectra: (2120 to 21008, 40 to 608), (280 to 2608, 240 to 2208), (60±808, 20±408) and (100±1208, 260 to 2408). In order to select the most appropriate area, we also observed the 2D spin-diffusion NMR spectrum of (AG)6A[1- 13C]G14A[1- 13C]G16(AG)7 as shown in Fig. 43. This spectrum depends on both the torsion angles of the 15th Ala and 16th Gly

C. Zhao, T. Asakura / Progress in Nuclear Magnetic Resonance Spectroscopy 39 (2001) 301±352

Fig. 45. The conformation of a repeated b-turn type II-like molecule as a model for silk I proposed here, there are intra-molecular hydrogen bonds between the carbonyl oxygen atom of the ith Gly residue and the amide hydrogen atom of the (i 1 3)th Ala residue [31].

residues, but re¯ects the torsion angles of the 16th Gly residue when the torsion angles of the 15th Ala residue are ®xed at (2608, 1308) as mentioned above. The calculated spectra are shown in Fig. 43 as a function of the f and c angles of the Gly residue for each 108 for four regions selected in Fig. 42. The region that was selected in the Ramachandran map is (f and c ) ˆ (608 to 808, 208 to 408). In order to obtain further information on the (f ,c ) angles of the Gly residue, the unit cell dimension along the chain axis, c value, of silk I was calculated. Thus far, three c values have been reported experimentally: Ê [121], 9.6 A Ê [124] and 8.88 A Ê [16]. Okuyama 9.08 A et al. [16] claimed that the larger value reported by Lotz and Keith [124] is due to a misinterpretation of the diffraction pattern, for which, Lotz and Keith assigned Ê to the plane (0 1 3) and then the re¯ection of 3.16 A determined the c value. However, this re¯ection should be assigned to the plane (1 1 2) of two layer lines. By considering the revised assignments, we have re-examined Okuyama's data [16]. The torsion angles …f ˆ 708; c ˆ 308† of the Gly residue along with f and c ˆ 2608 and 1308, respectively, of Ala residue satisfy the c Ê. value, 8.88 A

339

Fig. 46. Observed plots of DS/S0 against the corresponding NcTr values for REDOR experiments of (AG)6A[1- 13C]G14AG [ 15N]A17G(AG)6 to determine the distance between the 13CyO carbon of the 14th Gly residue and the 15N nitrogen of the 17th Ala residue. Continuous and dotted lines show the theoretical dephasing curves corresponding to the designated distance. The data marked by O are observed for the isotope-labeled compound without dilution of natural abundance (Ala±Gly)15 and those by K, for a mixture of an equivalent amount of the isotope-labeled compound and natural abundance (Ala±Gly)15. By comparing the REDOR data and the theoretical dephasing curve, the 13C± 15N Ê , which inter-atomic distance was determined to be 4.0 ^ 0.1 A Ê calculated for intra-molecular hydrogen agrees with the 4.0 A bond for the repeated b-turn type II-like structure [31].

In order to con®rm the torsion angles determined here, a REDOR experiment was performed. Fig. 44 shows two experimental 13C REDOR difference curves DS/S0 for samples (AG)6A[1- 13C]G14A[ 15N]G16(AG)7 and (AG)7[1- 13C]A15G[ 15N]A17G(AG)6. Solid lines show theoretical dephasing curves corresponding to the designated distances. By comparing the REDOR data with the theoretical dephasing curves, the 13C± 15N inter-atomic distances for the samples (AG)6A[1- 13C]G14A[ 15N]G16(AG)7 and (AG)7[1- 13C]A15G[ 15N]A17G(AG)6 were determined Ê , respectively. The broken lines to be 3.2 and 3.8 A Ê . These show that the experimental error is ^0.1 A distances are in good agreement with the predicted Ê , when the torsion angles of the values, 3.3 and 3.7 A Ala and Gly resides in the silk I structure are (2608, 1308) and (708, 308), respectively. The conformation of an (AG)15 chain with the silk I

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Fig. 47. Comparison of the observed (upper) and calculated (lower) WAXS patterns of (Ala±Gly)15 with silk I form. The WAXS pattern of silk I was simulated using the Insight II (release 4,0. OPT) diffraction pulldown in the crystall cell module [11].

form calculated with the torsion angles determined here is shown in Fig. 45. This is a repeated `b-turn type II structure' (the torsion angles for typical b-turn type II structures are; for the Ala residue, …f; c† ˆ …2608; 1208† and for the Gly residues …f; c† ˆ …808; 08† [15]. There is an intra-molecular hydrogen bond between the carbonyl oxygen atom of the ith Gly residue and the amide hydrogen atom of the (i 1 3)th Ala residue. The distance of this hydrogen bond between the carbonyl carbon and amide nitroÊ . REDOR offers the gen, was calculated to be 3.9 A 13 possibility to examine the C± 15N distances of the carbonyl carbon of the Gly residue and the amide nitrogen of the Ala residue, and thereby observe the formation of intra-molecular hydrogen bonds. Therefore, REDOR experiments were performed to determine the distance between the 13CyO carbon of the 14th Gly residue and the 15N nitrogen of the 17th Ala residue of (AG)6A[1- 13C]G14AG[ 15N]A17G(AG)6. The REDOR spectra were acquired on this isotope-labeled compound without dilution of natural abundance (AG)15 and also for a mixture of equivalent amounts of this isotope-labeled compound and the natural abundance (AG)15 in order to check whether or not these REDOR curves are unchanged as the labeled peptide molecule was diluted with unlabeled peptide. Although the latter curve has a relatively larger experimental error because of the overlapping of the

Fig. 48. Temperature dependence of solid-state 1H spin±lattice relaxation times, T1, of B. mori silk ®broin ®ber, ®lm, powder and the crystalline fraction powder, in dry system.

natural abundance Gly carbonyl peaks, the curves coincide with each other. The distance was deterÊ as shown in Fig. 46. Thus, mined to be 4.0 ^ 0.1 A Ê agrees with the the observed distance of 4.0 ^ 0.1 A Ê , the value calculated for the distance distance, 4.0 A of the intra-molecular hydrogen bond of the repeated b-turn type II-like structure. Anderson showed that the simulation of the WAXS pattern of silk I can be used to judge the validity of the various proposed silk I models [122]. Therefore, the experimental WAXS pattern of (AG)15 in the silk I form (Fig. 47, upper) was compared with the pattern simulated on the basis of both the torsion angles obtained here and the diffraction data (Fig. 47, lower): The unit cell is orthorhombic, and the space  group is P212121. The lattice constants are a ˆ 4:65 A;   b ˆ 14:24 A and c ˆ 8:88 A; a ˆ b ˆ g ˆ 908 [16]. The observed densities of the crystalline fraction of silk ®broin and of (AG)n, indicate that there are four repeated Ala±Gly units, in the unit cell. They form two 21-helix chains with anti-parallel arrangements in the unit cell. The agreement between the experimental

C. Zhao, T. Asakura / Progress in Nuclear Magnetic Resonance Spectroscopy 39 (2001) 301±352 Table 6 Relaxation time constants measured for N. clavipes dragline silk [59] Carbon

Proton T1r (ms)

Carbon T1 (s)

Ala Cb

7.9

Ala Ca Gly Ca Glu Cb,g (CyO)

8.5 7.5 7.9 7.8

0.18 (40%) 2 (60%) 12 9 4 20

and the calculated patterns is remarkably good (the R value was 13%) when the intensities of only six re¯ections are used [11]. Our model suggests that there are intermolecular hydrogen bonds whose direction runs perpendicular to the ®ber axis, and that there are intramolecular hydrogen bonds oriented roughly along the ®ber axis. Previous infrared dichroism experiments by Hirabayashi et al. suggested the existence of intermolecular hydrogen bonds perpendicular to the chain axis of the silk I form of B. mori silk ®broin [14]. Brack and Spach [125] have observed doublets in the amide absorption bands of the IR spectrum of poly(AG), suggesting the existence of two kinds of hydrogen bonds, which is in agreement with the present NMR results. The transition from silk I to silk II ®lms occurs at 2708C, indicating that the network of hydrogen bonds present in the structure of silk I makes it extremely stable to heat treatment. However, the silk I structure is rather unstable towards mechanical deformation [3,8,12,30]. It is highly probable that stretching of the silk I structure along the chain would disrupt the network of hydrogen bonds and convert the intramolecular hydrogen bonds to inter-molecular hydrogen bonds, to form the anti-parallel b-sheets that are characteristic of the silk II form. In the biological spinning process, the mature larva of the silkworm stretches the silk ®broin chain by performing head movements depicting the number `eight'. Finally, the model proposed here was compared with previous silk I models. Lotz and Cesari [19,20,124] have reported two sets of torsion angles for the (AG)n peptide; for Ala, the torsion angles are (2104.68, 112.28), and for Gly, they are (79.8, 49.78), or alternatively, for Ala, the values are (2124.58, 88.28) and for Gly, they are (249.88, 276.18). This model is not consistent with the 13C chemical shift

341

data of Ala in the silk I form, as pointed out previously [27,28] and it cannot explain the spin-diffusion and REDOR data obtained here. Likewise, Fossey's model [21] (where the (f ± c ) angles are (2808, 1508) for Ala and (21508, 808) for Gly) cannot account for the X-ray and electron diffraction data, as reported by Anderson [122]. 10. Dynamics of silk ®broin from NMR relaxation measurement and 2H NMR Andrew et al. studied the dynamical behavior of solid amino acids, polycrystalline peptides and tripeptides, a series of homo-polypeptides and polycrystalline proteins by 1H pulsed NMR [126±130]. Their results led to the conclusion that one of the most important motions in polypeptides and proteins giving rise to proton relaxation is the reorientation of the methyl groups in the side chains of alanine, isoleucine, methionine, threonine and valine residues. In addition, they discussed the contribution of water molecules in the protein structure to the relaxation above 250 K. Asakura et al. [131] reported the dynamical behavior of the B. mori silk ®broin chain and of absorbed water in silk ®ber, ®lm and powder as studied by 1H pulsed nuclear magnetic resonance. Fig. 48 shows the temperature dependence of the 1H spin±lattice relaxation times of B. mori silk ®broin ®ber (Silk II), ®lm (Silk I), powder (Silk II) and crystalline fraction of the silk ®broin prepared after chymotrypsin cleavage. The minimum relaxation time at 90 MHz occurs at about 270 to 2808C, independent of the sample. This relaxation comes from the intra-molecular motions because silk ®broin is essentially immobilized in the solid state. A temperature of 270 to 2808C, is approximately what is expected for a process involving reorientation of the methyl groups of the Ala residues. Segmental motions do not occur and only the rapid rotation of the methyl groups of the Ala residues is observed in the temperature of 2120± 1308C. This is independent of the conformation or form of the silk ®broin samples. Magnetization of dry silk ®broin created by the solid-echo method shows a single Gausssian decay, while two components are observed in the solid-echo signals of ®lms containing 6±10 w/w% water. An immobile component with a T2 value of 11 ms is attributed to silk

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Fig. 49. (A) Solid-state 30.5 MHz 2H spectrum of polycrystalline [3,3,3- 2H3] Ala with best ®t simulation (dotted line) using quadrupole splitting …vQ =2p† ˆ 40:4 kHz and FWHM ˆ 2100 Hz, with residual. (B) Spectrum of labeled N. clavipes dragline silk. Methyl component (90%) was simulated with …vQ =2p† ˆ 39:9 kHz; FWHM ˆ 2700 Hz. Non-methyl component (10%) was simulated with splitting of 120 kHz. (c) Spectrum of super-contracted silk. Methyl component (86%) was simulated as in (B); non-methyl component (14%) was simulated by a Gaussian distribution centered at zero frequency [136].

®broin, and the mobile component to bound water. The T2 of the latter varies from 50 to 200 ms, depending on the sample. The dynamical behavior of water trapped in the ®lm is discussed on the basis of these T2 values.

Table 6 shows the 1H T1r and 13C T1 relaxation time constants measured for N. clavipes dragline silk [59]. Each set of 1H T1r data was well ®tted by a single exponential. Backbone and side-chain carbons of glycine, alanine and glutamine have the same relaxation time constant of about 8 ms. Observation of a single T1r for all sites indicates that the size of the inhomogeneity present in the silk ®bers is limited to a few nanometers. The short relaxation time constant of 8 ms suggests that spin-diffusion results in the relaxation of all protons by a single ef®cient relaxation mechanism, probably the rotation of the Ala CH3 group. The 13C T1 values measured for dragline silk are of the same order of magnitude as those seen in silkworm silk [10]. The fact that glutamine CH2 groups relax more quickly than the Gly CH2 re¯ects the increased mobility of the side chains as compared to the backbone. It is clear that the carbonyl carbons relax very slowly, an expected result since they have no attached protons. The alanine CH relaxes almost as quickly as the Gly CH2, therefore, that the Ala CH relaxation rate is enhanced by the CH3 rotation in that residue. An excellent ®t to the intensities from the Ala CH3 was obtained by a two-component ®t in which 40% of the carbons have a relaxation time constant of 0.18 s, and 60% relax more slowly, with a T1 of 2 s. The observation of two T1 values indicates that Ala in dragline silk is present in two different motional environments. It is tempting to assign the slow-relaxing majority of the alanine carbons to the crystalline phase, while the remaining Ala present in the amorphous phase would be more mobile and relax faster. Deuterium solid-state NMR has frequently been used during the last decade to characterize the structure and dynamics of polymers, including proteins [132±135]. The advantage of 2H-NMR for studying deuterium-labeled polymers is that the line-shape and relaxation behavior are determined predominantly by the quadrupolar interaction, which can be up to 250 kHz. The coupling between the deuterium nuclear quadrupole moment and the electric ®eld gradient at the nucleus is a direct re¯ection of the local electron distribution in a particular bonding arrangement. Other interactions, such as the deuteron chemical shift or the deuteron±proton and deuteron±deuteron dipolar interactions, are about 100 times less in magnitude. 2H NMR spectroscopy is a powerful approach for examining the molecular motion of

C. Zhao, T. Asakura / Progress in Nuclear Magnetic Resonance Spectroscopy 39 (2001) 301±352

343

Fig. 50. Cartoon of the proposed model for the molecular arrangement of the alanine residues in a dragline silk ®ber. Highly oriented Ala-rich crystals of b-sheets (rectangles) and weakly oriented yet crystalline unaggregated sheets (canted sheet-like structures) are depicted in an amorphous glycine-rich matrix (curved lines). In reality, the Gly-rich matrix composes about 70% of the ®ber; in this drawing it has been largely suppressed for clarity [136].

polymers in the solid-state [132±135]. Different types of motion can be differentiated on the basis of their time scale and their geometry of exchange. The onedimensional quadrupole echo line shape of 2H NMR is especially sensitive to dynamics in the range of 10 28 , t c , 10 23 s, where t c is the motional correlation time. Within these limits, the 2H NMR line shape can be comprehensively analyzed in terms of wellknown models to yield the geometry and rate of segmental motion. The sensitivity of 2H NMR line shape and relaxation time to orientation and dynamics was used to selectively study the Ala-containing regions of silk. Jelinski et al. [136] presents that solid-state 2H nuclear magnetic resonance data from unoriented, oriented and super-contracted ®bers. Fig. 49(A) shows the 2H spectrum of polycrystalline [3,3,3- 2H 3] Ala which displays an axially symmetric powder pattern, narrowed by rapid methyl group reorientation. In the simulation of the line shape corrected for fall off of pulse power with frequency, the quadrupole coupling constant and the width of the Gaussian broading were the only adjustable parameters. Fig. 49(B) shows the 2H spectrum of unoriented dragline silk ®bers from spiders that had been fed deuterated alanine. The methyl deuterons were undergoing fast reorientation, as shown in the

Fig. 51. Solid-state 61.25 MHz 2H NMR powder spectra obtained from [3,3- 2H2]Ser-labeled B. mori (a) and S. c. ricini (b) silk ®broins. The respective line shapes (c) and (d) were simulated on the basis of a three-site jump model, showing one fast and one slow motional component each, plus a small contribution of 2HHO [80].

alanine. The spectrum also contains a small component with a splitting that is representative of static deuterons with perpendicular singularities at ^60 kHz. The spectrum could be satisfactorily

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Table 7 Powder pattern line shape simulation results of 2H quadrupole experiments for [3,3- 2H2]Ser-labeled B. mori and S. c. ricini [80] Component

Rate (Hz)

Occupancy

Libration (deg)

Fraction (%)

B. mori Fast Slow

1 £ 106 5 £ 103

(33:33:33) (90:5:5)

0 15

25 75

S. c. ricini Fast Slow

1 £ 106 5 £ 103

(33:33:33) (80:10:10)

0 15

22 78

simulated with the use of the sum of a methyl component that contributes 90% of the intensity and a static component that contributes 10% of the intensity. The repetition delay of 0.5 s in these spectra was insuf®cient to allow the non-methyl component to relax

Fig. 52. Observed (a, b) and simulated (c) solid-state 61.25 MHz 2H NMR spectra. (a) [3,3- 2H2]Tyr-labeled silk ®broin of B. mori; (b) [3,3- 2H2]Tyr-labeled silk ®broin of S. c. ricini; (c) spectral simulations based upon a three-site jump model. A 164 kHz quadrupole coupling constant was assumed and a rate constant of 10 3 Hz [138].

fully. A spectrum of fully relaxed component showed that 20% of the 2H in the silk was in sites other than the Ala methyl groups, which indicated that some scrambling of deuterons had occurred during the spider's metabolism. Fig. 49(C) shows the 2H spectrum of super-contracted silk that reveals that the methyl component is unchanged upon wetting. However, the static signal is replaced by a peak centered at zero frequency, which is typical for averaging of the quadrupolar coupling by fast isotropic reorientation. The static component is plasticized by water and must be in the amorphous domain. So the crystalline fraction of dragline silk consists of two types of Ala-rich regions, one that is highly oriented and one that is poorly oriented and less densely packed. A model for the molecularlevel structure of individual silk molecules and their arrangement in the ®bers is proposed in Fig. 50, a possible model for dragline silk is that some of the residues are present in a classical crystalline phase while the remainder are in protocrystals, possible preformed b sheet. The weakly oriented b sheets may account for the compressive strength of silk, as they provide reinforcement at a variety of angles. This hypothesis requires that individual protein molecules possess b-sheet conformation before ®ber formation. 2 H-NMR spectroscopy has also been applied to the structural analysis of B. mori and S. c. ricini silk ®broins [80,137±139]. The backbone structure of silk ®broin ®ber was shown to be an anti-parallel bsheet. The silk of the wild silkworm, S. c. ricini, has an amino acid composition and sequence rather different from B. mori. The primary structure of the silk ®broin from S. c. ricini has recently been determined by Yukuhiro et al. [140] and is very similar to the structure of silk ®broin from A. pernyi [43] (Fig. 5). This kind of silk ®broin is largely made up of repetitive

C. Zhao, T. Asakura / Progress in Nuclear Magnetic Resonance Spectroscopy 39 (2001) 301±352

Fig. 53. Observed (dotted line) and calculated (solid line) solid-state 61.25 MHz 2H NMR spectra of [3 0 ,5 0 - 2H2]Tyr-labeled B. mori and S. c. ricini silk ®broins. Spectra were obtained with the quadrupole echo pulse sequence with repetition times of 10s [138].

regions, each consisting of a polyalanine stretch of 10±14 residues followed by a Gly-rich sequence. Its total amino acid composition contains 33.2% Gly, 48.4% Ala, 5.5% Ser, 4.5% Tyr and 0.4% Val (Table 1). Fig. 51 shows the 2H solid-state NMR spectra of [3,3- 2H2]Ser-labeled B. mori(a) and S. c. ricini (b) silk ®broins [80,137]. The powder patterns (Fig.

345

51(a) and (b)) of the unoriented silk ®bers display no appreciable difference between B. mori and S. c. ricini. Both proteins give rise to the same set of quadrupole splitting of about 35.5 and 109.3 kHz. The inner component with a splitting of 35.5 kHz stems from Ser side chains undergoing rapid rotational motion, whereas the outer splitting represents comparatively immobile residues. A small central peak at zero frequency is attributed to residual 2HHO in the sample [131]. The line shapes of Fig. 51(c) and (d) were simulated using the `mxqet program' developed by Green®eld et al. [141], where the exchange rate and libration angle are calculated on the basis of appropriate dynamic models. The experimental spectra could properly be ®tted as a sum of three components, namely, the two types of motional distinct Ser side chains, plus a small 2HHO contribution with a Gaussian line shape. The best-®t parameters of these simulations are summarized in Table 7. As shown in Fig. 51(c), the spectrum for B. mori could be well simulated assuming that the slow motionally component contributes 75% and the rapid component 25% of the total intensity (see Table 7). The slow motional component in Fig. 51(c) was simulated assuming a discrete three-site jump of the Ser side chain, with unequal occupancies (90:5:5 ratio) and small librational amplitude. The fast component of B. mori satis®es a rapid three-site jump with equal occupancies. Likewise, the spectrum for S. c. ricini in Fig. 51(d) could be simulated with 78% corresponding to a slow three-site jump with unequal occupancies (80:10:10 ratio) and small librational amplitude with the remaining 22% arising from a rapid three-site jump. These results indicate that there are no signi®cant differences in the respective dynamic populations of B. mori and S. c. ricini silk ®broins, despite their unrelated amino acid sequences. Solid-state deuterium NMR ( 2H NMR) was also used to study the dynamics of the Tyr residues in silk ®broin from B. mori and S. c. ricini [138].

Table 8 Powder pattern line shape simulation results of 2H quadrupole±echo line shapes for [3 0 ,5 0 - 2H2]Tyr-labeled B. mori and S. c. ricini silk ®broin [138] Component

Fast Slow

B. mori

S. c. ricini

Rate (Hz)

Libration (deg)

Fraction (%)

Rate (Hz)

Libration (deg)

Fraction (%)

1 £ 106 1 £ 103

0 10

20 80

1 £ 107 1 £ 104

0 20

60 40

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Fig. 54. Expanded in vivo 67.8 MHz 13C solution NMR spectra of living 5-day-old, 5th instar larvae of B. mori (A) and P. c. ricini (B) after oral administration of 2.5 mg of [1- 13C]glucose. Peaks in spectra were identi®ed by comparison with those of standard samples [149].

Speci®cally deuterated cocoon silk was obtained by feeding silk worms with Tyr, labeled either at the Cb carbon ([3,3- 2H2]Tyr) or at the aromatic ring ([3 0 ,5 0 - 2H2]Tyr). The 2H NMR spectra of [3,3- 2H2]Tyr-labeled silk ®broins showed typical rigid powder patterns, indicating that there is essentially no motion about the Ca ±Cb bond axis, both in B. mori and S. c. ricini. Fig. 52 shows the experimental 2 H NMR spectra of [3,3- 2H2]Tyr-labeled B. mori(a) and S. c. ricini (b) silk ®broin, acquired with a recycle delay of 10 s. Fig. 52(c) demonstrates that the observed powder line shapes can be successfully simulated with an asymmetry parameter of h ˆ 0:00 and a quadrupole coupling constant of Q cc ˆ 164 kHz: On the basis of the model of a three-site jump around the Ca ±Cb bond, a very slow rate constant of 10 3 Hz was used. The agreement between

the observed (Fig. 52(a) and (b)) and simulated (Fig. 52(c)) spectra is good, indicating that the rotation about the Ca ±Cb bond axis can be considered as essentially static for both B. mori and S. c. ricini silk ®broins. As will be described below, a large proportion of Tyr rings undergo fast p -¯ips with a rate constant of $10 6 Hz. Therefore, the predominant side-chain motion of Tyr in silk ®broin is restricted to the phenolic ring. Fig. 53 shows the experimental (dotted line) and calculated (solid-line) 2H NMR spectra for [3 0 ,5 0 - 2H2]Tyr-labelled B. mori(a) and S. c. ricini (b) silk ®broins, with a recycle delay of 10 s. Because of their 2-fold symmetry, the phenolic side chains of Tyr can execute a p -¯ip motion about the Cb ±Cg bond, between two orientations of equal energy. Generally, any molecular motion reduces the quadrupole coupling to a time-averaged value

C. Zhao, T. Asakura / Progress in Nuclear Magnetic Resonance Spectroscopy 39 (2001) 301±352

347

Fig. 55. Expanded in vivo 67.8 MHz 13C solution NMR spectra of B. mori larva (A), and that of standard and extracted samples from the larva (B). (a) Gly at pH 11, (b) Gly at pH 6.7, (c) extract obtained from the midgut after oral administration of [2- 13C]Gly (pH 10), (d) hemolymph collected from the larva after the treatment of [2- 13C]Gly (pH 6.7) [34].

that is smaller than the rigid lattice constant. Thus, the small inner doublet with a splitting of 30 kHz, which is observed both for B. mori and S. c. ricini silk ®broins, is attributed to a fast p -¯ip motion of the phenolic ring. In contrast, the outer doublet with a splitting of 123 kHz corresponds to a slow motional component. The central peak at zero frequency is attributed to residual 2 HHO in the sample [131], which can be taken into account in the line-shape simulation by a Gaussian function. The fraction of the 2HHO component was assumed to be 0.8 and 2.5% of the total spectral intensity, for B. mori and S. c. ricini respectively. A comparison of the two different kinds of silk ®broin in Fig. 53(a) and (b) shows that the line shapes differ signi®cantly from one another, indicating that also the propensity for Tyr ring-¯ips must be different. The line shape could be consistently simulated with an asymmetry parameter of h ˆ 0:05 and a quadrupole coupling constant of Qcc ˆ 180:0 kHz: At least, two components were required to obtain a good ®t, and these two components can be considered as rigid and

as mobile on the deuterium NMR time scale. The rates obtained from the simulation analysis are compiled in Table 8 for B. mori and S. c. ricini silk ®broins. The dynamics of silk ®broin is also reveiwed by us [139]. 11. Silk ®broin synthesis in silkworm monitored by in vivo NMR It is important from both biological and technological viewpoints to clarify the biosynthetic pathway of silk in silkworm. For example, analysis of the 13C NMR spectrum showed that the carbons of labeled acetate administered to B. mori larvae are incorporated into the Ala residues of silk ®broin through malate and pyruvate [4]. Unique metabolic pathways of carbonyl-hydrates and amino acids are reported to exist in silkworm [142,143]. The ®rst demonstration of sorbitol in animals was in the diapause eggs of this insect [144]. In B. mori, free methylated amino acids accumulate in all stages of development, and the

348

C. Zhao, T. Asakura / Progress in Nuclear Magnetic Resonance Spectroscopy 39 (2001) 301±352 Table 9 Rate constants of [2- 13C]Gly transport in the B. mori larva [34]

Fig. 56. Relative area of 13C NMR peaks of glycine in the midgut (X), in the hemolymph (W), the silk ®broin (B) and citrate in the midgut (A) as a function of observation time. Relative area of each peak was determined by the peak simulation assuming a Lorentzian lineshape and corrected on the basis of 13C NMR relaxation times [34].

Fig. 57. Model of [2- 13C]Gly transport through the midgut into the silk ®broin (A) and four-compartment model for the Gly transport (B). Closed circles of C in the structural formula means 13C-labeled carbons. X and K are amount of 13C-labeled metabolites and in vivo rate constant for various transport of Gly or citrate [34].

Rate constant

£ 10 22 min 21

K1 K2 K3 K4 K5 K6 K7 K8

7.20 5.62 10.37 0.62 3.17 0.12 0.06 0.43

arginine degradation cascade reaction initiated by a speci®c endopeptidase, initiatorin, occurs for sperm maturation [145]. Alanine accumulated in silkworm eggs at the onset of diapause is converted to proline via glutamate during diapause [146]. Some of these metabolic pathways may be related to supply of amino acids for ®broin synthesis. NMR studies on living matter can be used for in vivo analysis of important metabolic pathways without use of chemical treatments [147,148]. The 13C NMR method has been used to examine the metabolism of [1- 13C]glucose and amino acid metabolism resulting from glucose metabolism in 5th instar larvae of the B. mori and S. c. ricini, in vivo and in vitro. Fig. 54 shows expanded in vivo 13C NMR spectra of living larvae of B. mori (A) and S. c. ricini (B) after oral administration of [1- 13C]glucose [149]. From spectra, after 30±40 min, the glucose signal was reduced, decrease of the b anomer being greater than that of the a anomer. At the same time, new signals appeared that were attributable to the C-1 carbons of glucose-6-phophate (G-6-P) and glucose-1-phosphate (G-1-P). This signal of G-1-P appeared later than that of G-6-P. After 240±250 min, during which the larva was not fed, the glucose signals disappeared and the C-1 carbon of trehalose increased. Asakura et al. reported the metabolic incorporation of [2- 13C]Gly into silk ®broin as studied by 13C NMR in vivo [34]. For the in vivo NMR measurement, 50 ml of 5% (w/v) [2- 13C]Gly in aqueous solution was given by oral administration to a 4-day-old 5th-instar larva after starvation for one day. Immediately, the larva was placed in a sample tube of 10 mm diameter and the NMR spectrum collected at 258C and without spinning the tube. Fig. 55 gives the expanded 13C NMR spectra of B. mori larva (A) and that of standard

C. Zhao, T. Asakura / Progress in Nuclear Magnetic Resonance Spectroscopy 39 (2001) 301±352

and extracted samples from the larva (B). The peak at 43.5 ppm is attributed to the a-carbon of the Gly residue of silk ®broin. In order to do further assignment, additional experiments were performed as follows. 13 C NMR spectra of the extracts from the midgut were observed after adjustment of pH to 10 corresponding to pH in the midgut of the 5th instar larva. Only two peaks at 44 and 46.5 ppm attributable to glycine-Ca and methylene carbons of citrate were observed. In addition, only one peak at 42.7 ppm attributable to the a-carbon of glycine was observed in 13C NMR spectra of hemolymph which was collected from the larvae after [2- 13C]Gly was injected into the hemolymph. And the change of relative area of each peak with time was determined in Fig. 56 where the peak simulation assuming a Lorentian shape was performed [34]. The correction of the peaks of the 13C labeled metabolites was performed by taking into account the NMR relaxation time. The peak intensity of Gly Ca carbon observed at pH 6.7 increased by 1.50 times after correction, Gly Ca at pH 6.7±1.50. Similarly, Gly Ca are at pH 10±1.88, Gly Ca of the residues of silk ®broin to 1.0 and citrate±CH2 ±at pH 10±1.42. The amounts of the Gly in the midgut rapidly decrease and that of citrate is low under these conditions. It is clear that the rapid transport of Gly from the midgut to the hemolymph is followed by incorporation into the silk ®broin. On the basis of the time-dependent peak intensities shown in Fig. 56, a model of transport of Gly in B. mori larva is proposed as shown in Fig. 57(A). In the midgut, a large portion of Gly is transported through the midgut membrane into the hemolymph, while a smaller portion is converted to citrate. The Gly in the hemolymph is absorbed into the posterior silk gland where it is incorporated into the silk ®broin. According to the compartment model as shown in Fig. 57(B), it is possible to discuss the kinetics of in vivo Gly transport. The rate constant of [2- 13C]Gly transport in B. mori larva obtained are listed in the Table 9. The rate constants are de®ned in Fig. 57(B), the relative areas of each 13C NMR peak are determined after the correction mentioned in materials and methods of Ref. [34]. As shown in Fig. 56, the solid lines that are calculated from the parameters listed in Table 9 agree well with the change of intensities. The value of K1/K5 is much larger than that of K2/K3, indicating that the rate of absorption through the midgut membrane into the

349

hemolymph is faster than the conversion from Gly to citrate in the midgut. Although K6, which represents the incorporation of Gly in the hemolymph into the ®broin in a relatively small value, another ¯ux of Gly in the hemolymph, K7, is much lower than K6. This suggests that the ef®ciency of incorporation of Gly into the silk ®broin is high in this stage of the larva. By assuming that the incorporation of Gly into silk ®broin in vitro follows ®rst order kinetics, the rate constant was determined by the curve-®tting as 7:4 £ 1024 min 21. This value was 63% of the corresponding value (K6) that was determined in vivo NMR of the silkworm larvae [34]. Acknowledgements TA acknowledges Prof. I. Ando at Tokyo Institute of Technology, Prof. H. Saito at Himeji Institute of Technology, Prof. T.A. Cross at Florida State University, Prof. A.S. Ulrich at Friedrich Schiller Universitaet Jena, Mr J.D. van Beek and Prof. B.H. Meier at ETH, Prof. T. Gullion at West Virginia University, Prof. M. Demura at Hokkaido University for stimulated discussion. TA also acknowledges support from the Program for Promotion of Basic Research Activities for Innovative Biosciences, Japan. References [1] Y. Tazima (Ed.), The Silkworm, Kodansha Scienti®c Books, Tokyo, 1978. [2] R.D.B. Fraser, T.P. MacRae, Conformations of Fibrous Proteins and Related Synthetic Polypeptides, Academic Press, New York, 1973. [3] T. Asakura, D.L. Kaplan, in: C.J. Arutzen (Ed.), Encyclopedia of Agriculture Science, vol. 4, Academic Press, London, 1994. [4] T. Asakura, JEOL News 23A (1987) 2. [5] R.D.B. Fraser, T.P. MacRae, F.H.C. Stewart, E. Suzuki, J. Mol. Biol. 11 (1965) 706. [6] R.D. Fraser, T.P. MacRae, F.H.C. Stewart, J. Mol. Biol. 19 (1966) 580. [7] H. Saito, Y. Iwanaga, R. Tabeta, M. Narita, T. Asakura, Chem. Lett. (1983) 427. [8] H. Saito, R. Tabeta, T. Asakura, Y. Iwanaga, A. Shoji, T. Ozaki, I. Ando, Macromolecules 17 (1984) 1405. [9] T. Asakura, Y. Watanabe, T. Itoh, Macromolecules 17 (1984) 2421. [10] H. Saito, M. Ishida, M. Yokoi, T. Asakura, Macromolecules 23 (1990) 83.

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