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Supercontracted spider dragline silk: a solid-state NMR study of the local structure
International Journal of Biological Macromolecules 24 (1999) 173 – 178
Supercontracted spider dragline silk: a solid-state NMR study of the local structure J.D. van Beek a,b, J. Ku¨mmerlen c, F. Vollrath d, B.H. Meier a,b,* a
NSR-Center for Molecular Structure, Design and Synthesis, Laboratory of Physical Chemistry, Uni6ersity of Nijmegen, Toernooi6eld, ED 6525, Nijmegen, The Netherlands b Laboratorium fu¨r Physikalische Chemie, ETH-Zentrum, 8092 Zu¨rich, Switzerland c Bayerisches Geo-Institut, Uni6ersita¨t Bayreuth, 95440 Bayreuth, Germany d Department of Zoology, Uni6ersitetsparken B135, DK 8000, Aarhus C, Denmark
Abstract The local structure of supercontracted dragline silk from the spider Nephila madagascariensis was investigated by solid-state nuclear magnetic resonance. Two-dimensional (2D) spin-diffusion experiments did not show any significant conformational changes in short-range order (and the secondary structure of the protein) upon supercontraction. Our results are in accordance with the proposal by Vollrath et al. (Proc R Soc London B 1996;263:147 – 151) that urea-supercontraction does not alter the local structure of spider dragline silk fundamentally. However, significant differences in the dynamics of the polypeptide chain upon supercontraction are detected at room temperature. At low temperature, these dynamics are frozen out. In addition, the role of the solvent (water) in the silk is investigated in Nephila edulis. Mobile water is detected at temperatures significantly below the freezing point of bulk water. © 1999 Elsevier Science B.V. All rights reserved. Keywords: Dragline silk; Nephila madagascariensis; NMR
1. Introduction Polymers are often able to absorb considerable quantities of (suitable) solvent when put into contact with it. The polymer network will rearrange to make the necessary space for the incoming solvent and the result of the process is a swollen gel-like phase with a volume that can be ten times larger than the dry network [1–3]. Some natural silks fibres contract upon contact with aqueous media [4,5]. During this reversible and repeatable process, called supercontraction, solvent is taken up by the silk, as in the swelling process described above. The fibre contracts to a fraction of its original length while the diameter increases correspondingly.
* Corresponding author. Present address: Laboratorium fu¨r Physikalische Chemie, ETH Zentrum, 8092 Zurich, Switzerland. E-mail address: [email protected] (B.H. Meier)
For most synthetic fibres supercontraction has been observed only under extreme conditions [4] but for silks aqueous solutions can suffice to, at least partially, bring about this process. Even though the outside appearance of silk drastically changes, X-ray diffraction studies have indicated that the structure of the crystalline parts is not affected significantly in the process of supercontraction [4]. However, evidence was found that the crystallizes reorient with respect to the fiber direction. Recently, it was proposed from microscopy data that a silk fiber consists of several tube-like layers of organization (of unknown relative dimensions) [5]. It was postulated that the structures observed in supercontracted silk match structures present in native spider dragline silk, even though a denaturing solvent was used to achieve supercontraction. In this paper we present further evidence for these proposals by using solid-state nuclear magnetic resonance NMR techniques.
0141-8130/99/$ - see front matter © 1999 Elsevier Science B.V. All rights reserved. PII: S 0 1 4 1 - 8 1 3 0 ( 9 8 ) 0 0 0 8 3 - X
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J.D. 6an Beek et al. / International Journal of Biological Macromolecules 24 (1999) 173–178
Fig. 1. Schematic orientation of two chemical shift tensors in the external magnetic field Bo. 2D spin-diffusion spectrum for (b) two single frequencies and for (c) many overlapping frequencies (powder pattern).
2. Local structure investigations by NMR NMR is capable of non-destructively investigating the local structure of compounds lacking long-range order. The NMR parameters most interesting in this context are the isotropic and anisotropic contribution to the chemical shift and the magnetic dipole interaction. The chemical shift delivers information about the electronic (or ‘chemical’) environment of the nucleus observed, in the present study 13C. For the interpretation of the experimental shift values empirical rules are usually employed [6], although ab initio calculations on smaller molecules become increasingly powerful [7]. The dipolar coupling is connected (by a simple relationship without adjustable parameters) to internuclear distances and orientations. Therefore, we will concentrate on the dipolar interaction in the following. The effective dipolar coupling constant between two nuclei i and j, d eff ij , is inversely proportional to the third power of the internuclear distance and also depends on the angle uij between the internuclear vector r ij and the external magnetic field B 0, d eff ij = −
m0 'gigj (3 cos2 uij −1) 4p r 3ij 2
(1)
Here, m0 denotes the vacuum permeability, gi the gyromagnetic ratio of nucleus i and ' Planck’s constant divided by 2p. The dipolar interaction can, in spin systems with isolated groups of spins, be observed directly as a splitting in the NMR spectrum, or it can be exploited as a mechanism for polarization transfer
between spins. The latter works also in complex manyspin systems where it is usually denoted as spin diffusion [8–10]. By measuring the spin-diffusion rate between two nuclei, the dipolar coupling constant can be determined. To do so, it is necessary to ‘label’ the source of the polarization, let the spin diffusion proceed, and then determine the destination of the polarization. Such a protocol can be realized in the framework of two-dimensional exchange spectroscopy [11,12], where the labelling and detection is done by using the inherent precession frequency of the spins, which in our case is given by the chemical shift. The chemical shift value depends on the orientation of the molecule-fixed coordinate system to the direction of the applied magnetic field. A macroscopically disordered sample will lead to a spread of resonance lines, i.e. a ‘powder pattern’. The spin-diffusion experiment (a particular kind of exchange experiment) proceeds as follows: initially, polarization is prepared on all 13C nuclei simultaneously. The polarization at each nucleus is labelled by the precession frequency of this nucleus in the so called evolution period. Then, the polarization is transferred to the neighboring spins in the mixing period by spin ‘flip-flop’ transitions caused by the dipolar interaction. Its destination is finally determined by the precession frequency of the nucleus where it ends up. In the two-dimensional (2D) spectrum, shown for a simple two spin example in Fig. 1b, the precession frequency of the source spin V1 is plotted against the frequency of the destination spin V2. If no polarization transfer took place, the resulting spectrum will be diagonal (V1 =V2).
J.D. 6an Beek et al. / International Journal of Biological Macromolecules 24 (1999) 173–178
However if polarization has been transferred between the two spins, the two frequencies in the v1 and v2 dimensions will differ and cross peaks (signals at V1 "V2) will appear in the 2D spectrum. For a powder sample the pattern will depend on the relative orientation of the chemical-shift tensor of the source and destination nuclei (Fig. 1c). Because the orientation of the chemical-shift tensors with respect to a molecule-fixed reference frame is usually known, or can be calculated, the relative orientation of neighboring molecular fragments can be determined even in the absence of macroscopic order. In the limit where the polarization has been spread equally among all spins of a certain domain of the material the spectral pattern will not change any more with longer mixing times. In such an quasi-equilibrium spectrum the distance information is lost and the exchange spectrum reflects solely the relative orientation of
175
the molecular segments observed [10]. The interpretation is then facilitated and iterative fitting of the spectra can reveal the local structure of the material. In previous work we have presented evidence that the local structure of both the alanine and glycine-rich domains in native spider dragline silk is partially ordered [13]. From two-dimensional proton-driven spin-diffusion experiments we proposed a simplified model in which the polyalanine segments adopt a highly ordered b-sheet structure, in accordance with other NMR measurements [14,15], and the glycine-rich domains tend to form 31-helical structures. As detailed in [13], 2D spin-diffusion experiments are quite sensitive and several other models, like a-helical, were shown to be incompatible with the experimental data. In this publication, the study is extended to supercontracted silk.
3. Materials and methods
Fig. 2. Schematic picture of the sample deformation due to the supercontraction process.
Fig. 3. Experimental 2D proton-driven spin-diffusion spectrum taken at l50 K of 1-13C alanine labeled spider dragline silk in the native state from N. madagascariensis with a mixing time of 10 s. (b) Corresponding spectrum at l50 K of a 1-13C glycine labeled sample. (c) Experimental 2D proton-driven spin-diffusion spectrum taken at 180 K of supercontracted 1-13C alanine labeled spider dragline silk from N. madagascariensis with 10 s mixing. (d) Corresponding spectrum at 180 K of a supercontracted 1-13C glycine labeled sample.
Silk was collected from Nephila madagascariensis and Nephila edulis at a speed of approximately 26 cm/min [16]. The spiders were kept at a low diet of Tenebrio mealworms supplemented with daily doses (starting a week before silking) of an aqueous amino acid solution. Supercontracted samples were obtained using 8 M urea using the procedure described by Vollrath et al. [5]. The silk samples consisted of about 40 mg of silk, approximately uniaxially oriented by winding the bundle around kevlar threads as shown in Fig. 2. The sample was kept oriented in the urea solution by pulling it using the Kevlar threads. After the supercontraction had occurred the silk was firmly wrapped around the fibers (Fig. 2). Excess solvent was removed with blotting paper after which the samples were let to dry for 2 h in open air before measurements were started. The outside appearance of the silk sample after supercontraction is quite different from that of the starting material: the samples had turned into a cement-like structure where the single fibers are not recognizable anymore. Mechanically, the sample was hard and not very elastic. Upon immersing it in plain water, the sample became gel-like again but it was not possible to regain the original shape of the sample: attempts to stretch the silk in the gel-like state by pulling the Kevlar strings resulted in damage to the material. NMR spectra were obtained on Bruker DMX 300 Avance and Bruker DMX 400 Avance spectrometers using a static 5 mm double-resonance probe (2D spin diffusion, 1H and 13C spectra) and a 4 mm triple-resonance probe (2D heteronuclear correlation) of the same manufacturer. Radio-frequency field strengths of typically 60 kHz have been used on both the carbon and proton channel. Contact times of 1.5 ms were used in all cross polarization experiments and the recycle delay was set to 4 s in all experiments.
J.D. 6an Beek et al. / International Journal of Biological Macromolecules 24 (1999) 173–178
176
Fig. 4. (a) Stack plot of two 1D 13C CP spectra taken at room temperature (RT) and 180 K (bottom line) just after (day 0) preparation of the supercontracted sample. (b) Corresponding spectra at 283 and 180 K (bottom line) on day 13 after the urea treatment.
All spectra have been processed using the MATLAB program [17]. The total signal intensity of the carbonyl region in the 2D spectra is normalized to 1000 for a digital resolution of 1.76 ppm per point. The levels in all contour plots shown here are absolute levels and are set at 0.025, 0.05, 0.075, etc. Such a procedure ensures that all contour levels from all plots can be compared directly.
4. Results and discussion We will first address the question of how the local structure (as seen by the spin-diffusion spectra) is influenced by supercontraction. In Fig. 3a and b the 2D spin-diffusion spectra of native 1-13C alanine and glycine-labeled unoriented spider dragline silk samples, taken at 150 K (from [13]), are shown. The corresponding spectra for supercontracted samples (Fig. 3c and d), taken at 180 K, show a qualitatively similar pattern. The mixing time for all spectra, and therefore the approximate spatial length-scale of the NMR structure determination, is the same for all spectra, namely 10 s. It is assumed that the spectra at this mixing time are quasiequilibrium spectra as discussed above [13]. Table 1 Second moments M2 for 1-13C tensors of Fig. 4 Spectrum
T (K)
M2 (rad2/s2)a
Fig. Fig. Fig. Fig.
298 180 283 180
1.26 1.53 1.49 1.48
4a, 4a, 4b, 4b,
top bottom top bottom
(4)×108 (6)×108 (5)×108 (2)×108
a Standard deviations have been estimated from the noise level in a signal free part of the spectrum.
The spectra of the native material have been interpreted by Ku¨mmerlen et al. [13] as follows: The spectrum of alanine remains almost diagonal and this can be attributed to a b-sheet structure of the alanine-rich domains of the fibroin where all the C= 0 bonds are approximately co-linear. The spectrum of glycine shows considerable off-diagonal intensity. A remarkably good fit of the experimental glycine data was obtained with a simple 31 helix model [13]. A description by a superposition of a disordered arrangement of the chains (‘random coil’) with a diagonal spectrum gave less satisfactory results but could not completely be excluded. In any case there was no evidence for a-helical structure elements. The pattern found for the alanine-labeled supercontracted sample is still highly diagonal, although slightly broader than for the native material, suggesting that the alanine-rich domains (known to be highly crystalline) retain their structure upon supercontraction. The amount of off-diagonal intensity in the supercontracted glycine spectrum (Fig. 3d) is significant although slightly lower than with the native silk (Fig. 3b). A quantitative comparison between the native and supercontracted patters is difficult however because the supercontracted samples have a different degree of isotopic labelling. Furthermore they are oriented macroscopically (although only slightly as represented in the 1D powder pattern, not shown) while the native samples were truly isotropic on a macroscopic scale. With these limitations in mind, it can be concluded that the local structure of the glycine-rich and alanine-rich domains in supercontracted and the native silks have a similar local structure. While our method may be insensitive to minor structural changes, substantial changes like the conversion of significant parts of the crystalline b sheets to a-helical structures are not supported by our data.
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177
Fig. 5. Stack plot of 1D 1H spectra taken at various temperatures for (a) native spider dragline silk from N. edulis and b) the same sample but supercontracted. Spectra were taken at 330, 300, 273, 253, 233, 210, 200 and 190 K (measured with the standard Bruker DMX 400 Avance equipment). The frequency scale is referenced to the position of the 1H line in bulk water.
In the following, we will address the question whether the mobility of the polypeptide backbone is altered by supercontraction. Fig. 4 shows 13C powder spectra from the super-contracted glycine-labeled sample taken at two temperatures at different time intervals after supercontraction. Rigid samples will lead to the well known chemical-shift tensor pattern of the carboxyl group. Mobility (fast on the NMR time scale of kHz) will lead to a partial averaging of this tensor and to a reduction of the second moment of the line. Just after preparing, the supercontracted sample shows significant mobility at room temperature as reflected by the line shape in Fig. 4a which can not be explained by a static chemical-shift tensor. The spectrum of the same sample at 180 K (Fig. 4a), shows no indications of dynamical processes anymore. Gradually solvent was
Fig. 6. Cross sections of two experimental 2D heteronuclear correlation experiments (WISE) performed under MAS (5100 and 5000 Hz spinning speed for the glycine and alanine sample, respectively) at room temperature.
allowed to evaporate from the supercontracted sample at room temperature. The spectra at room temperature gradually approached a static tensor line as seen in the spectrum of Fig. 4b which was taken after 13 days of exposure to ambient air. After 13 days the room-temperature and low-temperature spectra (283 and 180 K) are almost indistinguishable and very similar to the low-temperature spectrum taken at day 0. The second moment M2, defined as % I(v)·(v − 6)2 M2 =
v
(2) % I(v) v
was evaluated for all four spectra and the results are listed in Table 1. Here I(v) denotes the intensity in the 1D spectrum at frequency v (only the carboxylic resonance is taken into account), and v ¯ is the isotropic shift for the carboxylic group. Indeed the qualitative findings discussed above are represented in the numerical data. It can, therefore, be concluded that significant motion takes place in the freshly prepared supercontracted sample at room temperature. Whether this motion must be assigned to a local flexibility of the protein backbone or to motion of entire chain-segments which remain relatively rigid locally cannot be addressed by our measurements. As supercontraction is induced using a great amount of solvent we were interested in whether any changes could be seen in the proton spectrum before and after supercontraction. Fig. 5 shows the temperature dependences of the 1H spectrum for a sample of (a) native silk and (b) air-dried supercontracted silk from N. edulis. For these spectra the freshly prepared supercontracted sample was allowed to dry by blowing a small flow of nitrogen gas over the sample overnight. As can be seen in both plots a relatively mobile 1H component remains in both states of the silk at temperatures far
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below the freezing point of bulk water. The number of mobile protons is of the same order of magnitude for the two samples. The resonance frequency of the sharp component coincides, within the linewidth, with the one of bulk water and therefore we attribute this signal to water that is confined inside the silk. We have attempted to obtain further information of the location of the water within the fiber. To that end, we have performed an experiment that correlated the proton and carbon spectra of our sample (2D WISE) [18 – 20]. Fig. 6 shows a cross section along the 1H direction of a 2D MAS proton–carbon correlation experiment located, in the carbon dimension, at the centerband of the carboxylic resonance from the 1-13C alanine and glycine labeled supercontracted samples of dragline silk. These peaks reflect the spectrum of the protons in close contact with the amino acid. The lines are 54 and 32 kHz wide (FWHH) for the glycine and alanine samples, respectively. This is more than one order of magnitude broader than the water signal of Fig. 5 and is attributed to the protons of the amino acids themselves. The signal of the mobile protons is not detected probably because they are too mobile for cross polarization to be effective.
5. Conclusions Despite the huge impact of the supercontraction process on the outside appearance of the dragline silk no major structural changes are detected in the local structures as seen by spin-diffusion NMR upon supercontraction induced by urea. It is found that the observed degree of local order is slightly diminished compared to native silk but it is definitely not lost. These results could be explained, amongst others, with a tube-like organization of native and supercontracted spider dragline silk as proposed by Vollrath et al. [5]. One-dimensional 1H experiments, as a function of temperature, have shown mobile water molecules at temperatures far below the freezing point of free water. From that, we conclude that this water is incorporated into the silk.
.
Acknowledgements Technical support by H. Janssen and J. van Os, SON National HF–NMR Facility, University of Nijmegen and financial support by the Dutch Science Foundation (SON) are gratefully acknowledged. We thank H.B. Jørgensen and E. Rasmussen for silking the spiders.
References [1] Flory PJ. Principles of Polymer Chemistry. Cornell University Press, 1953. [2] Clark AH, Ross-Murphy SB. Adv Poll Sci 1987;83:57. [3] Tsuchida E, Abe K. Adv Poll Sci 1982;45:1. [4] Work RW, Morosoff N. Text Res J 1982;52:349 – 56. [5] Vollrath F, Holtet T, Thoegersen HC, Frische S. Proc R Soc London B 1996;263:147 – 51. [6] Mehring M. Principles of High Resolution NMR in Solids, 2nd edition. Berlin: Springer, 1983. [7] de Dios AC. J Prog NMR Spectrosc 1996;29:229 – 78. [8] Bloembergen N. Physica 1949;15:386. [9] Meier BH. Polarization transfer and spin diffusion in solid-state NMR. In: Warren WS, editor. Advances in Magnetic and Optical Resonance, vol. 18. New York: Academic Press, 1994:1–116. [10] Ernst M, Meier BH. Spin diffusion in solids. In: Ando I, Asakura T, editors. Solid State NMR of Polymers. Amsterdam: Elsevier, 1998. [11] Jeener J, Meier BH, Bachmann P, Ernst RR. J Chem Phys 1979;71:4546. [12] Ernst RR, Bodenhausen G, Wokaun A. Principles of Nuclear Magnetic Resonance in One and Two Dimensions. Oxford: Clarendon Press, 1987. [13] Ku¨mmerlen J, van Beek JD, Vollrath F, Meier BH. Macromolecules 1996;29:2920 – 8. [14] Simmons A, Ray E, Jelinski LW. Macromolecules 1994;27:5235 – 7. [15] Simmons AH, Michal CA, Jelinski LW. Science 1996;271:84–7. [16] Work RW, Emerson PD. J Arachnol 1982;10:1 – 10. [17] MATLAB is a commercial program for numerical manipulations and visualizations from the Mathworks Inc., Natick, MA. [18] Caravatti P, Deli JA, Bodenhausen G, Ernst RR. J Am Chem Soc 1982;104:5506. [19] Schmidt-Rohr K, Clauss J, Spiess HW. Macromolecules 1992;25:3273 – 7. [20] Clauss J, Schmidt-Rohr K, Adam A, Boeffel C, Spiess HW. Macromolecules 1992;25:5208 – 14.
Supercontracted spider dragline silk: a solid-state NMR study of the local structure J.D. van Beek a,b, J. Ku¨mmerlen c, F. Vollrath d, B.H. Meier a,b,* a
NSR-Center for Molecular Structure, Design and Synthesis, Laboratory of Physical Chemistry, Uni6ersity of Nijmegen, Toernooi6eld, ED 6525, Nijmegen, The Netherlands b Laboratorium fu¨r Physikalische Chemie, ETH-Zentrum, 8092 Zu¨rich, Switzerland c Bayerisches Geo-Institut, Uni6ersita¨t Bayreuth, 95440 Bayreuth, Germany d Department of Zoology, Uni6ersitetsparken B135, DK 8000, Aarhus C, Denmark
Abstract The local structure of supercontracted dragline silk from the spider Nephila madagascariensis was investigated by solid-state nuclear magnetic resonance. Two-dimensional (2D) spin-diffusion experiments did not show any significant conformational changes in short-range order (and the secondary structure of the protein) upon supercontraction. Our results are in accordance with the proposal by Vollrath et al. (Proc R Soc London B 1996;263:147 – 151) that urea-supercontraction does not alter the local structure of spider dragline silk fundamentally. However, significant differences in the dynamics of the polypeptide chain upon supercontraction are detected at room temperature. At low temperature, these dynamics are frozen out. In addition, the role of the solvent (water) in the silk is investigated in Nephila edulis. Mobile water is detected at temperatures significantly below the freezing point of bulk water. © 1999 Elsevier Science B.V. All rights reserved. Keywords: Dragline silk; Nephila madagascariensis; NMR
1. Introduction Polymers are often able to absorb considerable quantities of (suitable) solvent when put into contact with it. The polymer network will rearrange to make the necessary space for the incoming solvent and the result of the process is a swollen gel-like phase with a volume that can be ten times larger than the dry network [1–3]. Some natural silks fibres contract upon contact with aqueous media [4,5]. During this reversible and repeatable process, called supercontraction, solvent is taken up by the silk, as in the swelling process described above. The fibre contracts to a fraction of its original length while the diameter increases correspondingly.
* Corresponding author. Present address: Laboratorium fu¨r Physikalische Chemie, ETH Zentrum, 8092 Zurich, Switzerland. E-mail address: [email protected] (B.H. Meier)
For most synthetic fibres supercontraction has been observed only under extreme conditions [4] but for silks aqueous solutions can suffice to, at least partially, bring about this process. Even though the outside appearance of silk drastically changes, X-ray diffraction studies have indicated that the structure of the crystalline parts is not affected significantly in the process of supercontraction [4]. However, evidence was found that the crystallizes reorient with respect to the fiber direction. Recently, it was proposed from microscopy data that a silk fiber consists of several tube-like layers of organization (of unknown relative dimensions) [5]. It was postulated that the structures observed in supercontracted silk match structures present in native spider dragline silk, even though a denaturing solvent was used to achieve supercontraction. In this paper we present further evidence for these proposals by using solid-state nuclear magnetic resonance NMR techniques.
0141-8130/99/$ - see front matter © 1999 Elsevier Science B.V. All rights reserved. PII: S 0 1 4 1 - 8 1 3 0 ( 9 8 ) 0 0 0 8 3 - X
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J.D. 6an Beek et al. / International Journal of Biological Macromolecules 24 (1999) 173–178
Fig. 1. Schematic orientation of two chemical shift tensors in the external magnetic field Bo. 2D spin-diffusion spectrum for (b) two single frequencies and for (c) many overlapping frequencies (powder pattern).
2. Local structure investigations by NMR NMR is capable of non-destructively investigating the local structure of compounds lacking long-range order. The NMR parameters most interesting in this context are the isotropic and anisotropic contribution to the chemical shift and the magnetic dipole interaction. The chemical shift delivers information about the electronic (or ‘chemical’) environment of the nucleus observed, in the present study 13C. For the interpretation of the experimental shift values empirical rules are usually employed [6], although ab initio calculations on smaller molecules become increasingly powerful [7]. The dipolar coupling is connected (by a simple relationship without adjustable parameters) to internuclear distances and orientations. Therefore, we will concentrate on the dipolar interaction in the following. The effective dipolar coupling constant between two nuclei i and j, d eff ij , is inversely proportional to the third power of the internuclear distance and also depends on the angle uij between the internuclear vector r ij and the external magnetic field B 0, d eff ij = −
m0 'gigj (3 cos2 uij −1) 4p r 3ij 2
(1)
Here, m0 denotes the vacuum permeability, gi the gyromagnetic ratio of nucleus i and ' Planck’s constant divided by 2p. The dipolar interaction can, in spin systems with isolated groups of spins, be observed directly as a splitting in the NMR spectrum, or it can be exploited as a mechanism for polarization transfer
between spins. The latter works also in complex manyspin systems where it is usually denoted as spin diffusion [8–10]. By measuring the spin-diffusion rate between two nuclei, the dipolar coupling constant can be determined. To do so, it is necessary to ‘label’ the source of the polarization, let the spin diffusion proceed, and then determine the destination of the polarization. Such a protocol can be realized in the framework of two-dimensional exchange spectroscopy [11,12], where the labelling and detection is done by using the inherent precession frequency of the spins, which in our case is given by the chemical shift. The chemical shift value depends on the orientation of the molecule-fixed coordinate system to the direction of the applied magnetic field. A macroscopically disordered sample will lead to a spread of resonance lines, i.e. a ‘powder pattern’. The spin-diffusion experiment (a particular kind of exchange experiment) proceeds as follows: initially, polarization is prepared on all 13C nuclei simultaneously. The polarization at each nucleus is labelled by the precession frequency of this nucleus in the so called evolution period. Then, the polarization is transferred to the neighboring spins in the mixing period by spin ‘flip-flop’ transitions caused by the dipolar interaction. Its destination is finally determined by the precession frequency of the nucleus where it ends up. In the two-dimensional (2D) spectrum, shown for a simple two spin example in Fig. 1b, the precession frequency of the source spin V1 is plotted against the frequency of the destination spin V2. If no polarization transfer took place, the resulting spectrum will be diagonal (V1 =V2).
J.D. 6an Beek et al. / International Journal of Biological Macromolecules 24 (1999) 173–178
However if polarization has been transferred between the two spins, the two frequencies in the v1 and v2 dimensions will differ and cross peaks (signals at V1 "V2) will appear in the 2D spectrum. For a powder sample the pattern will depend on the relative orientation of the chemical-shift tensor of the source and destination nuclei (Fig. 1c). Because the orientation of the chemical-shift tensors with respect to a molecule-fixed reference frame is usually known, or can be calculated, the relative orientation of neighboring molecular fragments can be determined even in the absence of macroscopic order. In the limit where the polarization has been spread equally among all spins of a certain domain of the material the spectral pattern will not change any more with longer mixing times. In such an quasi-equilibrium spectrum the distance information is lost and the exchange spectrum reflects solely the relative orientation of
175
the molecular segments observed [10]. The interpretation is then facilitated and iterative fitting of the spectra can reveal the local structure of the material. In previous work we have presented evidence that the local structure of both the alanine and glycine-rich domains in native spider dragline silk is partially ordered [13]. From two-dimensional proton-driven spin-diffusion experiments we proposed a simplified model in which the polyalanine segments adopt a highly ordered b-sheet structure, in accordance with other NMR measurements [14,15], and the glycine-rich domains tend to form 31-helical structures. As detailed in [13], 2D spin-diffusion experiments are quite sensitive and several other models, like a-helical, were shown to be incompatible with the experimental data. In this publication, the study is extended to supercontracted silk.
3. Materials and methods
Fig. 2. Schematic picture of the sample deformation due to the supercontraction process.
Fig. 3. Experimental 2D proton-driven spin-diffusion spectrum taken at l50 K of 1-13C alanine labeled spider dragline silk in the native state from N. madagascariensis with a mixing time of 10 s. (b) Corresponding spectrum at l50 K of a 1-13C glycine labeled sample. (c) Experimental 2D proton-driven spin-diffusion spectrum taken at 180 K of supercontracted 1-13C alanine labeled spider dragline silk from N. madagascariensis with 10 s mixing. (d) Corresponding spectrum at 180 K of a supercontracted 1-13C glycine labeled sample.
Silk was collected from Nephila madagascariensis and Nephila edulis at a speed of approximately 26 cm/min [16]. The spiders were kept at a low diet of Tenebrio mealworms supplemented with daily doses (starting a week before silking) of an aqueous amino acid solution. Supercontracted samples were obtained using 8 M urea using the procedure described by Vollrath et al. [5]. The silk samples consisted of about 40 mg of silk, approximately uniaxially oriented by winding the bundle around kevlar threads as shown in Fig. 2. The sample was kept oriented in the urea solution by pulling it using the Kevlar threads. After the supercontraction had occurred the silk was firmly wrapped around the fibers (Fig. 2). Excess solvent was removed with blotting paper after which the samples were let to dry for 2 h in open air before measurements were started. The outside appearance of the silk sample after supercontraction is quite different from that of the starting material: the samples had turned into a cement-like structure where the single fibers are not recognizable anymore. Mechanically, the sample was hard and not very elastic. Upon immersing it in plain water, the sample became gel-like again but it was not possible to regain the original shape of the sample: attempts to stretch the silk in the gel-like state by pulling the Kevlar strings resulted in damage to the material. NMR spectra were obtained on Bruker DMX 300 Avance and Bruker DMX 400 Avance spectrometers using a static 5 mm double-resonance probe (2D spin diffusion, 1H and 13C spectra) and a 4 mm triple-resonance probe (2D heteronuclear correlation) of the same manufacturer. Radio-frequency field strengths of typically 60 kHz have been used on both the carbon and proton channel. Contact times of 1.5 ms were used in all cross polarization experiments and the recycle delay was set to 4 s in all experiments.
J.D. 6an Beek et al. / International Journal of Biological Macromolecules 24 (1999) 173–178
176
Fig. 4. (a) Stack plot of two 1D 13C CP spectra taken at room temperature (RT) and 180 K (bottom line) just after (day 0) preparation of the supercontracted sample. (b) Corresponding spectra at 283 and 180 K (bottom line) on day 13 after the urea treatment.
All spectra have been processed using the MATLAB program [17]. The total signal intensity of the carbonyl region in the 2D spectra is normalized to 1000 for a digital resolution of 1.76 ppm per point. The levels in all contour plots shown here are absolute levels and are set at 0.025, 0.05, 0.075, etc. Such a procedure ensures that all contour levels from all plots can be compared directly.
4. Results and discussion We will first address the question of how the local structure (as seen by the spin-diffusion spectra) is influenced by supercontraction. In Fig. 3a and b the 2D spin-diffusion spectra of native 1-13C alanine and glycine-labeled unoriented spider dragline silk samples, taken at 150 K (from [13]), are shown. The corresponding spectra for supercontracted samples (Fig. 3c and d), taken at 180 K, show a qualitatively similar pattern. The mixing time for all spectra, and therefore the approximate spatial length-scale of the NMR structure determination, is the same for all spectra, namely 10 s. It is assumed that the spectra at this mixing time are quasiequilibrium spectra as discussed above [13]. Table 1 Second moments M2 for 1-13C tensors of Fig. 4 Spectrum
T (K)
M2 (rad2/s2)a
Fig. Fig. Fig. Fig.
298 180 283 180
1.26 1.53 1.49 1.48
4a, 4a, 4b, 4b,
top bottom top bottom
(4)×108 (6)×108 (5)×108 (2)×108
a Standard deviations have been estimated from the noise level in a signal free part of the spectrum.
The spectra of the native material have been interpreted by Ku¨mmerlen et al. [13] as follows: The spectrum of alanine remains almost diagonal and this can be attributed to a b-sheet structure of the alanine-rich domains of the fibroin where all the C= 0 bonds are approximately co-linear. The spectrum of glycine shows considerable off-diagonal intensity. A remarkably good fit of the experimental glycine data was obtained with a simple 31 helix model [13]. A description by a superposition of a disordered arrangement of the chains (‘random coil’) with a diagonal spectrum gave less satisfactory results but could not completely be excluded. In any case there was no evidence for a-helical structure elements. The pattern found for the alanine-labeled supercontracted sample is still highly diagonal, although slightly broader than for the native material, suggesting that the alanine-rich domains (known to be highly crystalline) retain their structure upon supercontraction. The amount of off-diagonal intensity in the supercontracted glycine spectrum (Fig. 3d) is significant although slightly lower than with the native silk (Fig. 3b). A quantitative comparison between the native and supercontracted patters is difficult however because the supercontracted samples have a different degree of isotopic labelling. Furthermore they are oriented macroscopically (although only slightly as represented in the 1D powder pattern, not shown) while the native samples were truly isotropic on a macroscopic scale. With these limitations in mind, it can be concluded that the local structure of the glycine-rich and alanine-rich domains in supercontracted and the native silks have a similar local structure. While our method may be insensitive to minor structural changes, substantial changes like the conversion of significant parts of the crystalline b sheets to a-helical structures are not supported by our data.
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Fig. 5. Stack plot of 1D 1H spectra taken at various temperatures for (a) native spider dragline silk from N. edulis and b) the same sample but supercontracted. Spectra were taken at 330, 300, 273, 253, 233, 210, 200 and 190 K (measured with the standard Bruker DMX 400 Avance equipment). The frequency scale is referenced to the position of the 1H line in bulk water.
In the following, we will address the question whether the mobility of the polypeptide backbone is altered by supercontraction. Fig. 4 shows 13C powder spectra from the super-contracted glycine-labeled sample taken at two temperatures at different time intervals after supercontraction. Rigid samples will lead to the well known chemical-shift tensor pattern of the carboxyl group. Mobility (fast on the NMR time scale of kHz) will lead to a partial averaging of this tensor and to a reduction of the second moment of the line. Just after preparing, the supercontracted sample shows significant mobility at room temperature as reflected by the line shape in Fig. 4a which can not be explained by a static chemical-shift tensor. The spectrum of the same sample at 180 K (Fig. 4a), shows no indications of dynamical processes anymore. Gradually solvent was
Fig. 6. Cross sections of two experimental 2D heteronuclear correlation experiments (WISE) performed under MAS (5100 and 5000 Hz spinning speed for the glycine and alanine sample, respectively) at room temperature.
allowed to evaporate from the supercontracted sample at room temperature. The spectra at room temperature gradually approached a static tensor line as seen in the spectrum of Fig. 4b which was taken after 13 days of exposure to ambient air. After 13 days the room-temperature and low-temperature spectra (283 and 180 K) are almost indistinguishable and very similar to the low-temperature spectrum taken at day 0. The second moment M2, defined as % I(v)·(v − 6)2 M2 =
v
(2) % I(v) v
was evaluated for all four spectra and the results are listed in Table 1. Here I(v) denotes the intensity in the 1D spectrum at frequency v (only the carboxylic resonance is taken into account), and v ¯ is the isotropic shift for the carboxylic group. Indeed the qualitative findings discussed above are represented in the numerical data. It can, therefore, be concluded that significant motion takes place in the freshly prepared supercontracted sample at room temperature. Whether this motion must be assigned to a local flexibility of the protein backbone or to motion of entire chain-segments which remain relatively rigid locally cannot be addressed by our measurements. As supercontraction is induced using a great amount of solvent we were interested in whether any changes could be seen in the proton spectrum before and after supercontraction. Fig. 5 shows the temperature dependences of the 1H spectrum for a sample of (a) native silk and (b) air-dried supercontracted silk from N. edulis. For these spectra the freshly prepared supercontracted sample was allowed to dry by blowing a small flow of nitrogen gas over the sample overnight. As can be seen in both plots a relatively mobile 1H component remains in both states of the silk at temperatures far
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below the freezing point of bulk water. The number of mobile protons is of the same order of magnitude for the two samples. The resonance frequency of the sharp component coincides, within the linewidth, with the one of bulk water and therefore we attribute this signal to water that is confined inside the silk. We have attempted to obtain further information of the location of the water within the fiber. To that end, we have performed an experiment that correlated the proton and carbon spectra of our sample (2D WISE) [18 – 20]. Fig. 6 shows a cross section along the 1H direction of a 2D MAS proton–carbon correlation experiment located, in the carbon dimension, at the centerband of the carboxylic resonance from the 1-13C alanine and glycine labeled supercontracted samples of dragline silk. These peaks reflect the spectrum of the protons in close contact with the amino acid. The lines are 54 and 32 kHz wide (FWHH) for the glycine and alanine samples, respectively. This is more than one order of magnitude broader than the water signal of Fig. 5 and is attributed to the protons of the amino acids themselves. The signal of the mobile protons is not detected probably because they are too mobile for cross polarization to be effective.
5. Conclusions Despite the huge impact of the supercontraction process on the outside appearance of the dragline silk no major structural changes are detected in the local structures as seen by spin-diffusion NMR upon supercontraction induced by urea. It is found that the observed degree of local order is slightly diminished compared to native silk but it is definitely not lost. These results could be explained, amongst others, with a tube-like organization of native and supercontracted spider dragline silk as proposed by Vollrath et al. [5]. One-dimensional 1H experiments, as a function of temperature, have shown mobile water molecules at temperatures far below the freezing point of free water. From that, we conclude that this water is incorporated into the silk.
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Acknowledgements Technical support by H. Janssen and J. van Os, SON National HF–NMR Facility, University of Nijmegen and financial support by the Dutch Science Foundation (SON) are gratefully acknowledged. We thank H.B. Jørgensen and E. Rasmussen for silking the spiders.
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