[30] Solution nuclear magnetic resonance characterization of peptide folding

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input provided by Drs. Paul Johnston, Richard Vandlen, and John Burnier, all of Genentech, Inc., South San Francisco, CA. The work carried out at the Howard Florey Institute was supported by an Institute Blockgrant from the NationalHealth and MedicalResearch Council of Australia.

[30] S o l u t i o n N u c l e a r M a g n e t i c R e s o n a n c e Characterization of Peptide Folding By KEVIN H. MAYO

Introduction The advent of solid-phase peptide synthesis has opened the door to various fields in biochemistry: understanding protein folding and folding pathways; protein fragment activity studies; de novo design of novel peptides; peptide ligand-receptor interactions; and drug discovery. To more fully understand how any peptide behaves or functions, conformational analyses are required. Two spectroscopic methods, nuclear magnetic resonance (NMR) and circular dichroism (CD), have been called on most frequently to assess the type and degree of peptide folding. This article, while focused primarily on solution NMR characterization of peptide folding, cannot exclude information from CD studies, which are most often presented along with complementary NMR experiments. For this reason, some reference will be made to various CD studies on peptides as related to NMR-based investigations. The usually convoluted CD trace represents a weighted conformational average over all backbone positions, whereas N M R has the capability of reporting weighted averages at specific backbone positions. Although this article is not meant to be a reference compendium for all NMR studies that have been done on peptides, pertinent examples from the literature are discussed. The following sections are presented to give the reader a brief overview of the use of N M R in analyzing conformational populations of peptides in solution. Peptides in Water The term structured protein or peptide refers to that overall conformational state which exists as a single, compact fold or as a family of very closely related folds. The "folded" state exists because the polypeptide chain has sufficient thermodynamic "stability" from various internal interactions to maintain a "tight knit" conformation. In aqueous solution, however, any short linear peptide (without sufficient "poly" in it, i.e., less than

METHODS IN ENZYMOLOGY, VOL. 289

Copyright © 1997 by Academic Press All rights of reproduction in any form reserved. 0076-6879/97 $25.00

[30]

SOLUTIONNMR OF PEPTIDEFOLDING Helix

Turn

Unfolded state

647

Sheet

Collapsed state

["Random coil" state]

FiG. 1. Simple scheme of peptide folding equilibria showing a few possible backbone conformations as discussed in the text. about 40 to 50 residues) and lacking sufficient structurally stabilizing forces, exists as a highly fluctuating distribution of conformational states as depicted schematically in Fig. 1. Here, the terms "unfolded," "collapsed," "helix," "turn," and "/3 sheet" have been used to indicate some of the possible "folding" contributions in this conformational ensemble. Whereas the later three are self-explanatory, the former two are least clear. The "collapsed" state is perhaps best described as a hydrophobically mediated association of side chains resulting in or not resulting in some clear structural elements; at one extreme would lie the "molten globule" state a which has a collapsed hydrophobic core and considerable secondary structure but lacks a defined, fixed tertiary structure. A true, fully "unfolded" state would be one where all residues are maximally exposed to solvent; this state probably does not actually exist in water since some folding, however transient, would tend to screen hydrophobic residues from water (i.e., some hydrophobic collapse would occur. 2 This of course depends on the amino acid sequence, composition, and length. In general, equilibria are shifted toward what is most often refered to in the literature as the " r a n d o m coil" state (shown in Fig. 1 as some combination of unfolded and collapsed state), such that the population of any one "folded" state is usually small. O. B. Ptitsyn and G. V. Misotnov, in "Conformations and Forces in Protein Folding" (B. T. Nail and K. A. Dill, eds.). American Association for the Advancement of Science, Washington, D.C., 1991. z V. A. Daragan, E. E. Ilyina, C. G. Fields, G. B. Fields, and K. H. Mayo, Protein Sci. 6, 355 (1997).

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Early attempts to identify secondary structure formation within linear peptides were generally unsuccessful (see, e.g., Epand and Scheraga3). This lack of evidence led to the premature conclusion that long-range interactions occurring within proteins were absolutely essential for the formation of secondary structure and that short linear peptides were largely unstructured in aqueous solution. A major reason for the dearth of observations of secondary structure in short peptides in aqueous solution was the absence of a sensitive probe for small populations of structured conformers within a conformational ensemble. However, one exception did exist early on: the C-peptide of ribonuclease A (residues 1-13) was found to populate helical conformations to approximately 30% (as assayed by using CD) in aqueous solution at 1.7°.4 Since that time, many peptides have been found by using NMR and CD to populate a variety of "folded" conformations in aqueous solution, in dynamic equilibrium with "random coil" forms (reviewed by Dyson and WrightS). The presence of small fractions of ordered structure in equilibrium with predominantly random coil conformations, however, is not only difficult to detect but also difficult to define. Does the detection of, for example, 5% helical structure mean that 5% of the population is in a fully helical conformation, or are there regions of fluctuating structure in different regions which average to that value? Because populations of "folded" structure are usually very small and the lifetimes of individual states within this conformational ensemble are short on the NMR chemical shift time scale, NMR resonances average, with the most populous "random coil" state being weighted the most. This gives rise to the so-called random coil NMR spectrum of a peptide as exemplified in Fig. 2 (bottom). Here, a 33-residue peptide has been dissolved in 90% H20/10% D20, pH 6.3, 40°. 1H r e s o n a n c e s are little dispersed from those of very short peptides as defined in tetrapeptides for all 20 common amino acid residues. 6'7 Backbone NH resonances fall between 7.5 and 8.7 ppm; no a H resonances are observed downfield of the water resonance, and resonances arising from side-chain methyls all overlap at around 1 ppm. This spectrum is in contrast to the 1H NMR spectrum of another 33-residue peptide (/3pep-4 from Mayo et al. 8) shown in Fig. 2 (top). There, many backbone NH and a H resonances are downfield shifted 3 R. M. Epand and H. A. Scheraga, Biochemistry 7, 2864 (1968). 4 j. E. Brown and W. A. Klee, Biochemistry 10, 470 (1971). 5 H. J. Dyson and P. E. Wright, Annu. Rev. Biophys. Biophys. Chem. 20, 519 (1991). 6 A. Bundi and K. Wiithrich, Biopolymers 18, 285 (1979). 7 K. W0thrich, " N M R of Proteins and Nucleic Acids." Wiley (Interscience), New York, 1986. 8 K. H. Mayo, E. Ilyina, and H. Park, Protein Sci. 5, 1301 (1996).

[30]

SOLUTIONNMR oF PEPTIDEFOLDING

649

?

-5

P

"~-10 ' 200 i 220 240 Wavelength(nm) I~~VJ t I upfield" ~ II

~H aromatic

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'

'

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'

,

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'

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,

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.

,

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.

.

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.

,

FIG. 2. 1H NMR spectra (600 MHz) of two peptide 33-mers (5 mM peptide in 10 mM potassium phosphate, pH 6.3, 313 K). NMR data were acquired on a Bruker AMX-600 spectrometer. Insets show CD traces of three peptide 33-mers as discussed in the text; CD data were acquired on a Jasco JA-710 spectropolarimeter.

by up to about 1 ppm from their random coil positions, and methyl resonances are upfield ring current shifted resulting from proximity to aromatic side chains. On first inspection, this relatively good chemical dispersion suggests compact folding. Furthermore, downfield NH and a H resonance shifting is one indication of the specifics of folding, namely, /3-sheet folding.

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SPECIALIZED APPLICATIONS

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Wishart et al. 9 have established a simple formalism for deriving helix and fl-sheet populations on the basis of the deviation of observed 1H chemical shifts from a "random coil" reference set. When viewed over a number of residues in a given sequence, downfield shifts indicate/3-sheet formation while upfield shifts indicate helix formation. Because an observed chemical shift represents a linear combination of random coil and more folded states, the degree of deviation can be used to discern relative "folded" populations. In the present case (Fig. 2, top), greater than 90% sheet formation is indicated. More specific structural details result from a complete analysis of various multidimensional NMR data sets. 7 For this peptide 33-mer, as for most peptides studied by NMR, the usual approach consists of acquiring 1H homonuclear two-dimensional (2D) NMR COSY, TOCSY, and NOESY data. COSY (correlated spectroscopy) and TOCSY (total correlated spectroscopy) experiments (and variations thereof) correlate resonances whose protons are through-bond coupled, whereas NOESY (nuclear Overhauser effect spectroscopy) experiments provide information on which protons are through-space dipole-dipole coupled. The power of the nuclear Overhauser effect (NOE) in terms of structural elucidation lies in the fact that an NOE is inversely proportional to the sixth power of the internuclear distance. For short linear peptides which usually exist in solution as an equilibrium of highly fluctuating conformational states, the internuclear distance is considerably more variable than in a compactly folded protein, and derivation of accurate internuclear distances is complicated by the fact that the observed NOE represents some unknown distance-of-closest-approach weighted average. Moreover, what is often less appreciated is that the NOE is also directly proportional to a function which is dependent on internuclear motions. In this respect, interpretation of NOE data, particularly for peptides, should be approached with caution. For peptide conformational analyses, however, NOE data may provide a qualitative picture of preferred conformational populations. As with any peptide/protein conformational analysis, sequence-specific resonance assignments must be done first. Here, COSY and TOCSY experiments are used initially to help identify or to group spin-coupled resonances into a particular amino acid residue type. This is often referred to as "spin system identification." For example, a residue having a short side chain often can be distinguished from one having a long side chain, or a polar residue from a nonpolar one. Ser, Thr, Gly, and Ala have a more easily distinguishable spin system, whereas Tyr, His, Trp, Phe, Asp, and Asn residues and Glu, Gln, and Met residues usually show characteristic a l l 9D. S. Wishart,B. D. Sykes,and F. M. Richards,Biochemistry 3L 1647 (1992).

[30]

SOLUTIONNMR OF PEPTIDEFOLDING

651

/~H2 and o~H-/3H2-TH2 patterns, respectively. Longer chain aliphatic and Lys spin systems are often problematic to discern. Spin system identification is exemplified in Fig. 3, which shows a "fingerprint" region from a TOCSY spectrum of a 27-residue peptide, y27, derived from the C terminus of the human fibrinogen y chain. 1° The sequence runs from residues 385 to 411 as Lys-Ile-Ile-Pro-Phe-Asn-Arg-Leu-Thr-Ile-Gly-Glu-Gly-Gln-Gln-HisHis-Leu-Gly-Gly-Ala-Lys-Gln-Ala-Gly-Asp-Val. For orientation in this TOCSY contour plot, the NH resonance domain runs horizontally and the o~H and side-chain proton resonance domain runs vertically. For any 2DN M R experiment, the diagonal (not shown) represents the normal N M R spectrum, and off-diagonal cross-peaks shown here identify which resonances are coupled. In this TOCSY experiment, therefore, cross-peaks identify which NH's are through-bond coupled to which o~H's and sidechain proton resonances. Although the intent of this section is not to provide an exhaustive spectral analysis, some key points can be made. Notice that the five Gly o~H2 resonances all fall around 4 ppm, and residues with an otH-/3H2 fragment have their/3H2 resonances falling between 2.5 and 3.5 ppm, with Asn and Asp resonances usually lying more upfield. 7 The/3HzTH2 protons of the four Glu/Gln spin systems resonate with a characteristic pattern between 1.9 and 2.5 ppm. The two Ala spin systems are identified by a relatively intense /3H3 resonance coupled directly to the respective c~H resonance, and even the single Thr-393 can be picked out by the highly downfield shifted/3H (4.2 ppm) being coupled to an upfield/3H3. Ile and Val spin systems often may be identified through their more upfield shifted methyl resonances and /3H resonances around 1.8 to 2.2 ppm; Val /3H resonances are usually the more downfield ones. Sequence-specific resonance assignments labeled in Fig. 3 resulted from subsequent analysis of a NOESY spectrum (Fig. 4) which was used to identify sequential spin systems, usually by the presence of an N O E between the a H of one residue and the NH of a sequential neighbor. Spin systems are thereby placed in a sequence that can then be compared to the known chemical sequence for sequence-specific resonance assignments. In this example (Fig. 4), TOCSY a H - N H cross-peaks are boxed in, and lines connect (i, i + 1) a H - N H NOEs. A good starting point is usually one of the unambiguous spin systems (e.g., Ala, Gly, Thr). For example, starting with either Ala spin system, one can unambiguously connect six spin systems as Gly-Ala-Xxx-(Glu/Gln)-Ala-Gly, where Xxx is probably a long sidechain spin system and Glu/Gln cannot be distinguished. When compared with the known amino acid sequence given above, this hexapeptide fragment can be unambiguously assigned as Gly4°4-Ala4°5-Lys4°6-Gln4°7-Ala 4°8-

u~K. H. Mayo, C. Burke, J. N. Lindon, and M. Kloczewiak,Biochemistry 29, 3277 (1990).

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.

.

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.

.

.

.

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[30]

SOLUTION N M R OF PEPTIDE FOLDING

397

395

409

404

653

,~

4.0 385 ,f

394

E ~

393

402 388 &

389

387

I

8.7

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FIG. 4. Backbone N H - a H fingerprint region from a NOESY contour plot. TOCSY crosspeaks have been boxed in, and lines connect spin systems in the sequence. Sample conditions are the same as in legend for Fig. 3.

Gly 4°9. Other spin system fragments were likewise identified and assigned. The unique Thr spin system had to be Thr-393 and was used to identify the sequence Pro-388 a H to Gly-395 NH. Because all Gly all2 (but not NH) resonances overlap somewhat, ambiguity arises and the sequence must start again from a different point. The I1e-387 to Pro-388 connection is made between the Ile-387 otH and the Pro-388 o°1-I2(upfield, not shown) since Pro has no NH. In this way, the complete amino acid sequence can be assigned and traced as shown in Fig. 4. Having completed sequence-specific resonance assignments, preferred conformational populations, if present, may be identified from further analysis of NOESY data as well as from analyses of coupling constants, back-

FI6. 3.2D NMR TOCSY contour plot of the NH-otH and upfield region for a 27-residue peptide derived from 3' chain of human fibrinogen, 3,27. The peptide concentration was 5 mM in 20 mM potassium phosphate in 90% H20/10% D20.

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bone NH temperature factors, and the presence of long-lived NHs. In a compactly folded protein, identification of numerous long-range NOEs, that is, greater than i, i + 3,4,5, usually can be made; coupling constants range from about 4 Hz (helix) to 9 Hz (sheet); and NH's form hydrogen bonds to yield low NH temperature factors and long-lived NHs. For peptides in water alone, however, the situation is very different. Long-range NOEs are usually not observed; instead, mostly i, i + 1 and possibly i, i + 2 NOEs are evident; coupling constants most often average to between 6 and 8 Hz (conformationally uninformative), and hydrogen bonds are weak, if present at all, resulting in higher NH temperature factors and few longlived NH's. 3,27 peptide was no exception, and a few weak i, i + 2 otH-NH NOEs provided some evidence for turn formation, while a series of mostly weak i, i + 1 N H - N H NOEs (Fig. 5) suggested transient helix formation from Iie-394 to Leu-402. For further information in performing conforma-

0..

8.8 8.8

8.3

7.8

PPM FIG. 5. N H - N H region from a N O E S Y contour plot. Sample conditions are the same as in legend for Fig. 3.

[30]

SOLUTION N M R OF PEPTIDE FOLDING

655

tional analyses using NMR, the reader is referred to texts by Wtithrich,7 Clore and Gronenborn, 11 and Evans. 12 Of further interest to understanding folding of peptides is a comparison of NMR and CD data. For example, both peptide 33-mers discussed above give CD traces (insets to Fig. 2) indicative of significant/3-sheet conformation (labeled with/3), 13 whereas NMR spectra allow one to differentiate between compact (Fig. 2, top) and noncompact (Fig. 2, bottom) folding. The CD trace for the NMR "random coil" peptide (Fig. 2, bottom), however, does indicate significant/3-strand secondary structure (labeled with /3). In this case, any apparent/3-sheet structure must be highly transient. Moreover, another peptide 33-mer gives both a "random coil" CD trace (labeled with " R " in Fig. 2, inset) and NMR spectrum (not shown; essentially the same as in Fig. 2, bottom); here, no preferred secondary or tertiary structure can be deduced. What makes peptides like these 33-mers fold or not fold is an area of intense investigation in the peptide folding field. Peptides have been used as models for initial folding events. Fragments derived from a structured protein may mimic the "unfolded" state and allow analysis of local interactions while eliminating the complication of tertiary interactions. 5'14-24 Initial folding of a complete protein sequence does not necessarily give rise to structures that are retained in the final folded form of the protein; examples exist of structures found in peptides that are not seen in the intact protein. 25 However, it does appear that the protein primary sequence codes for secondary structure found in the native 1~G. M. Clore and A. M. Gronenborn, "NMR of Proteins." CRC Press, Boca Raton, Florida, 1993. lz j. N. S. Evans, "Biomolecular NMR Spectroscopy." Oxford Univ. Press, Oxford, 1995. ~3G. Fasman, ed., "Circular Dichroism and the Conformational Analysis of Biomolecules." Plenum, New York, 1996. 14 p. E. Wright, H. J. Dyson, and R. A. Lerner, Biochemistry 27, 7167 (1988). 15 D. Shortle and A. K. Meeker, Biochemistry 28, 936 (1989). 16 p. S. Kim and R. L. Baldwin, Annu. Rev. Biochem. 59, 631 (1990). 17H. J. Dyson, G. Merutka, J. P. Waltho, R. A. Lerner, and P. E. Wright, J. Mol. BioL 226, 795 (1992). ~s H. J. Dyson, J. R. Sayre, G. Merutka, H.-C. Shin, R. A. Lerner, and P. E. Wright, J. Mol. Biol. 226, 819 (1993). 19j. Sancho and A. R. Fersht, J. Mol. Biol. 224, 741 (1992). 20 y. Kuroda, T. Nakai, and T. Ohkubo, J. Mol. Biol. 236, 862 (1994). 21 H. Shin, G. Merutka, J. Waltho, P. Wright, and H. Dyson, Biochemistry 32, 6348 (1993). 22 H. Shin, G. Merutka, J. Waltho, L. Tennant, J. Dyson, and P. Wright, Biochemistry 32, 6356 (1993). 23j. Waltho, V. Feher, G. Merutka, H. Dyson, and P. Wright, Biochemistry 32, 6337 (1993). 24 A. D. Kippen, J. Sancho, and A. Fersht, Biochemistry 33, 3778 (1994). 25 H. J. Dyson, K. J. Cross, R. A. Houghten, I. A. Wilson, P. E. Wright, and R. A. Lerner, Nature (London) 318, 480 (1985).

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folded protein; this was demonstrated with NMR by using a series of peptide fragments derived from two different protein folds: the peptides derived from the/3-sheet protein plastocyanin showed conformational preferences predominantly for extended/3 structure in solution, TMwhereas those derived from the four-helix bundle protein myohemerythrin showed considerable propensity for helical structures. I7 Problems with conformational equilibria always arise. Most often these tend to be associated with the lack of observable NMR parameters. Because of rapid conformational averaging and the shift in equilibrium to the "random coil" state (Fig. 1), chemical shifts are structurally uninformative, J coupling constants average to 6-8 Hz, and NOEs are significantly weakened. In addition, observed NOEs represent some weight average over a number of structural states, complicating conformational analysis. Moreover, NH temperature factors and hydrogen/deuteron exchange data, often used to deduce hydrogen bonding in a structured protein/peptide, are less useful and more difficult to interpret with "unstructured" peptides. A further problem arising from conformational equilibria may occur when various folding interactions are quasi-stable and lifetimes of some conformations fall into an intermediate or slow exchange regime on the NMR chemical shift time scale. This gives rise to line broadening and/or to individual resonances for each long-lived exchange state. In general, the latter is not a problem with short linear peptides which remain monomeric; however, the presence of aggregation can induce any number of unwanted species. Furthermore, cis-trans-proline isomerization, which occurs slowly on the NMR chemical shift time scale,7 can present serious problems particularly in peptides where conformational stability is low compared to a structured protein. This conformational "freedom" tends to shift the cistrans equilibrium toward the cis state at the expense of the normally more populated trans state. Two sets of resonances are then observed for each isomer; chemical shift separation is greatest for residues sequentially flanking the proline. It should also be mentioned that even though numerous NMR structural studies have been done using 15N- and 13C-labeled proteins, most peptide NMR investigations have been limited to 1H NMR techniques. The main reasons for this are (1) the usual small size of the peptide and (2) the cost of aSN- and 13C-labeled and derivatized amino acid residues required for synthesis. In future studies on peptides, more use probably will be made of selective isotopic enrichment in peptides; some advantages would be increased sensitivity to relatively small conformational populations in a highly overlapped 1H NMR spectrum and possible characterization in the "unfolded" state. 2,26 ~H NMR characterization of peptide folding has been 26T. M. Logan,E. T. Olejniczak,R. X. Xu, and S. W. Fesik,J. Biomol. NMR 3, 225 (1993).

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SOLUTION N M R OF PEPTIDE FOLDING

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done on peptides with conformational populations from turns, helices, coiled coils, fl hairpins,/3 sheets, to collagen triple helices. The following sections exemplify these various structural types. Helices a n d T u r n s

Reverse turns are determined by short-range interactions and limit the conformational space available to a polypeptide chain. It has long been recognized that certain amino acid sequences have a high probability of being part of a turn conformation in proteins, 27'28and this has more recently been shown by NMR and CD to be true also for peptides in aqueous solution. 29 In some of these short linear peptides, characteristic /~-turn NOEs have been observed: N H - N H between turn positions 3 and 4, and a H - N H between turn positions 2 and 4. The weakness of these NOEs indicates partial folding. Several NMR studies done on cell adhesion-promoting Arg-Gly-Asp-containing hexapeptides found evidence for "nested" fl turns (see Mayo et al. 3° and references therein). Furthermore, Stradley et al.3~ found that when turn-type NOEs are observed in short peptides, they usually represent an equilibrium among various types of turns, particularly type-I (III) and type-II. Turns have been implicated in the folding of helices: the structure known as "nascent helix" formed in short peptides in aqueous solution 32 consists of a series of interlinked turnlike conformations that readily form helix in the presence of solvents such as trifluoroethanol or in the intact protein. A "nascent helix" is characterized by a continuous series of N H - N H NOEs and weak or absent i, i + 2,3,4 NOEs. If a sequence forms turnlike structures in solution as an initiating event in the folding process, then it is likely that the structure will persist in longer fragments containing the sequence. By investigating short linear peptides derived from specific structural units in the native protein, that is, helix, turn, or fl sheet/turn regions, conformational preferences in early folding intermediates may be postulated under equilibrium conditions. When solvent conditions are the same as native folding conditions, it may be argued that relatively "stable" conformations probably do exist early on in the protein folding conformational ensemble. 27 G. D. Rose, L. M. Gierasch, and J. A. Smith, Adv. Protein Chem. 37, 1 (1985). 28 p. y . Chou and G. D. Fasman, Adv. Enzymol. 47, 45 (1978). 29 H. J. Dyson, M. Rance, R. A. Houghten, R. A. Lerner, and P. E. Wright, 3. MoL Biol. 201, 161 (1988). 3o K. H. Mayo, F. Fan, M. P. Beavers, A. Eckardt, P. Keane, W. J. Hoekstra, and P. AndradeGordon, Biochim. Biophys. Acta 1296, 95 (1996). 31 S. J. Stradley, J. Rizo, M. D. Bruch, A. N. Stroup, and L. M. Gierasch, Biopolymers 29, 263 (1990). 32 H. J. Dyson, M. Rance, R. A. Houghten, P. E. Wright, and R. A. Lerner, J. Mol. Biol. 201, 201 (1988).

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That being the case, it m a y be possible to identify structures which either form first or at least are stable intermediates. These potential "intermediates" m a y or m a y not remain as part of the native protein structure. The approach of using synthetic peptides derived f r o m structural units in native proteins has been increasingly exploited. K e m m i n k and Creighton 33 studied the conformational properties of seven overlapping peptides, 9 to 16 residues long, that comprise the entire primary structure of bovine pancreatic trypsin inhibitor (BPTI). The peptides are largely disordered but have somewhat different average conformational propensities, in line with various secondary structure predictions. R e d u c e d B P T I appears to be approximately the sum of its individual peptides. Several local interactions involving aromatic rings of side chains interacting with groups nearby in the primary structure have b e e n identified and verified by replacing the responsible side chains. Some of these interactions were found to play a role in folding of reduced BPTI. Wright and co-workers 2a-23 have u n d e r t a k e n an extensive N M R study of peptide fragments corresponding to regions in myoglobin that have b e e n implicated in early folding events. Waltho e t aL 23 and Shin e t al. 21 studied the conformational preferences of peptides corresponding to the G and H helices and of the intervening sequence, termed the G - H turn. Optical methods failed to detect helical structure, TM but small conformational preferences for helical structure were found by NMR. 35'36 Waltho e t aL 23 and Shin e t al. 21 demonstrated that a peptide corresponding to the entire H helix contains a significant population of helical structure in solution as a consequence of the removal of nonhelical residues at the C terminus and the additional residues at the N terminus that form part of the H helix in the native protein. This study indicates that formation of stable structure depends to some extent on where the peptide is "clipped." Related to this, Munoz e t aL 37 showed that inclusion of a "hydrophobic-staple" at the end of a helix p r o m o t e s increased stability; in an 18-mer, they noted n u m e r o u s longer-range helix N O E s in about half of the p r o p o s e d helix. N M R also has been used to establish the existence of N- and C-terminal capping interactions in peptides. 38,39 These primarily 33j. Kemmink and T. E. Creighton, J. MoL Biol. 234, 861 (1993). 34jo Hermans and D. Puett, Biopolymers 10, 895 (1971). 35j. p. Waltho, V. A. Feher, R. A. Lerner, and P. E. Wright, F E B S Lett. 250, 400 (1989). 36j. p. Waltho, V. A. Feher, and P. E. Wright, in "Current Research in Protein Chemistry" (J. J. Villafranca, ed.), p. 283. Academic Press, San Diego, 1990. 37V. Munoz, F. J. Blanco, and L. Serrano, Nat. Struct. Biol. 2, 380 (1995). 38p. C. Lyu, D. E. Wemmer, H. X. Zhou, R. J. Pinker, and N. R. Kallenbach, Biochemistry 32, 421 (1993). 39H. X. Zhou, P. C. Lyu, D. E. Wemmer, and N. R. Kallenbach, J. A m . Chem. Soc. 116, 1139 (1994).

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SOLUTION N M R OF PEPTIDE FOLDING

659

arise from hydrogen-bonding interactions of a polar side chain (e.g., Asn, Gin, Set) with a backbone carbonyl or amine group. Such interactions also appear to play an important role in helix stabilization in short linear peptides. Although native capping interactions have been identified in short linear peptides, nonnative capping interactions have also been observed. 4°,41 fl S h e e t s

In terms of studying short peptides with fl-sheet folding tendencies, the situation differs as attempts to observe isolated/3 sheets have usually been hampered by the strong tendency of these peptides to aggregate and by a general lack of requisite longer range interactions that dictate a more stable /3-sheet fold. The smallest fl-sheet unit is one in which two antiparallel/3 strands are joined by a/3 turn. This is often referred to as a/3 hairpin. Generally, these too are not stable in water and often aggregate. Some exceptions, however, have been reported. Blanco et al. 42 studied a short linear peptide derived from the B1 domain of protein G and found that it folds to some extent (40%) into a/3-hairpin conformation in water. By replacing a turn in a/3-hairpin peptide derived from Tendamistat with the sequence NPDG (highest predicted score to adopt a type I/3 turn), Blanco et aL 43 showed that that peptide also folds into a/3 hairpin in water. These same turn residues have also been found to stabilize a nonnative hairpin conformation in a peptide derived from the N-terminal/3-hairpin of ubiquitin. 44More recently, Ramirez-Alvarado et aL 45 designed a novel dodecapeptide which was found by NMR to adopt a 30% populated/3-hairpin structure in water. Although interstrand NOEs consistent with/3-sheet structure were observed (4-9 otH-o~H; 3-10 NH-NH; 5-8 NH-NH), a series of relatively strong N H - N H NOEs also were observed from residue 5 to residue 12, suggesting the additional presence of nascent helix conformation32 in relatively fast exchange with the/3-hairpin population. This raises an interesting point that when dealing with short peptides, NOEs representing multiple conformations may be observed, and caution should be exercised when interpreting data. Two well-characterized examples of/3-sheet formation in somewhat longer peptides have been found in a disulfide-bridged heterodimeric model 40 E. Ilyina, R. Milius, and K. Mayo, Biochemistry 33, 13436 (1994). 41 B. Odaert, F. Baleux, T. Huynh-Dinh, J. M. Neumann, and A. Sanson, Biochemistry 34, 12820 (1995). 42 F. Blanco, G. Rivas, and L. Serrano, Nat. Struct. Biol. 1, 584 (1994). 43 F. J. Blanco, G. Rivas, and L. Serrano, J. Am. Chem. Soc. 115, 5887 (1993). 44 M. S. Searle, D. H. Williams, and L. C. Packman, Nat. Struct. Biol. 2, 999 (1995). 45 M. Ramirez-Alvarado, F. J. Blanco, and L. Serrano, Nat. Struct. Biol. 3, 604 (1996).

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of a protein folding domain 46 and a zinc-finger peptide where a two-stranded amphipathic/3 sheet is stabilized by the folding an amphipathic helix onto it. 47 In both cases, the presence of a short length of/3 sheet was established by interstrand NOEs. Several other peptides have been proposed to form /3 sheets on the basis of N M R data, 48-5° but for none of these are the data completely convincing because no cross-sheet NOEs were observed. In a protein dissection study, one peptide 20-mer derived from the /3 sheet of platelet factor-4 (PF4) was shown by N M R to have a nativelike turn conformation that is stabilized by hydrophobic side-chain interactions from flanking/3-strand sequences. 4° Moreover, some nonnative helixlike conformational populations were noted in one of the/3 strands. In related studies, peptide 33-mers comprising the full/3-sheet domain from PF451 and from interleukin-8 (IL-8) 8 showed native-like, compact/3-sheet sandwich folding. Collagen M o d e l Peptides

Although most N M R peptide conformational studies have been done on peptides derived from globular proteins containing more traditional a-helical,/3-sheet, and turn motifs, triple helices do represent another major structural motif. The triple helix is the main structural unit found in all collagens and in a variety of other proteins and is generally composed of the amino acid sequence repeat (X-Y-Gly)n. In fibrillar collagens, this repeat can extend to about 1000 residues long. Nonglobular, fibrillar proteins like most collagens are difficult to work with from a structural perspective. For example, most do not crystallize, obviating X-ray diffraction analysis, and size, solubility, and gelation generally preclude solution N M R studies. Alternatively, one can work with considerably shorter fragments of the triple helix, for example, peptides generated by synthetic techniques. T o date, various triple-helical model peptides such as (Pro-Pro-Gly)n and (Pro-Hyp-Gly)n have been studied by using NMR. Mayo et al.52 studied the 1H N M R solution conformation of a nonconsensus collagen peptide Gly-Val-Lys-Gly-Asp-Lys-Gly-Asn-Pro-Gly-Trp-ProGly-Ala-Pro-Tyr (called peptide IV-HI), derived from the protein sequence of human collagen type IV, triple helix domain residues 1263-1277. The N M R data on peptide IV-H1 indicated significant/3-turn populations cen46T. G. Oas and P. S. Kim, Nature (London) 336, 42 (1988). 47M. S. Lee, G. Gippert, K. Y. Soman, D. A. Case, and P. E. Wright, Science 245, 635 (1989). 48R. Saffrichm,H. R. Kalbitzer, H. Bodenmuller, P. Muhn, R. Pipkorn, and H. C. Schaller, Biochim. Biophys. Acta 997, 144 (1985). 49S. C. J. Sumner and J. A. Ferretti, FEBS Lett. 253, 117 (1989). 50j. B. Vaughn and J. W. Taylor, Biochim. Biophys. Acta 999, 135 (1989). 51E. Ilyina and K. H. Mayo, Biochem. Z 306, 407 (1995). 52K. H. Mayo, D. Parra-Diaz, J. B. McCarthy,and M. Chelberg,Biochemistry 30, 8251 (1991).

[30]

SOLUTION N M R OF PEPTIDE FOLDING

661

tered at Lys3-Gly4, LysS-Gly6, Pro9-Gly 1°, and Pro12-Gly13, and a C-terminal y-turn within the Ala14-ProlS-Tyr I6 sequence. Although this multiple turn conformation may or may not be found in native collagen, it is one structural possibility for a nonconsensus collagen sequence, many of which are found in native collagen. By using NMR, CD, and cross-linking, Hoppe et al. 53 showed that the structure of a 35-residue collagen-type sequence from lung surfactant protein D consists of a triple-stranded parallel a-helical bundle in a nonstaggered, and extremely strong, noncovalent association. This type of association between three polypeptide chains may represent a common structural feature immediately following the C-terminal end of the triplehelical region of collagenous proteins. Li et al. 54 have used 1H and 15N NMR to structurally characterize two consensus collagen triple-helical peptides: (Pro-Hyp-Gly)10, which is considered to be the most stable triple helix model peptide, and (Pro-HypGly)3Ile-Thr-Gly-Ala-Arg-Gly-Leu-Ala-Gly-Pro-Hyp-Gly (Pro-Hyp-Gly)3 (called T3-785), designed to model an imino acid poor region of collagen. 15N enrichment has been selective at specific Ala and Gly positions. Both peptides associated as trimers with melting temperatures of 60° for (ProHyp-Gly)10 and 25 ° for T3-785. In T3-785, with nonrepeating Xxx-Yyy-Gly units incorporated in the sequence, the three chains of the homotrimer were distinguishable in NMR spectra. The solution conformations of (ProHyp-Gly)10 and T3-785 were similar to the model derived from X-ray fiber diffraction data, with the closely packed central residues of the three chains showing a one-residue stagger of the three parallel chains. In a related NMR study, Long et al. 55 designed a set of four (Pro-HypGly)i0 peptides to model the effect of (Gly-X-Y)n repeat interruptions on triple-helix formation, stability, and folding. An interruption in the middle of this stable triple-helical peptide still allowed formation of trimers in solution, but with decreased stability which depended on the type of interruption. A Gly substitution or an Ala insertion were least disruptive, whereas a Gly deletion was the most destabilizing and a Hyp deletion was intermediate. Their results suggested that such interruptions in the collagen repeat pattern affected both triple-helix and monomer conformations. These studies provided a general approach to analyzing the percent triple helix composition in a collagen peptide. By measuring the loss of monomer resonance intensity and the increase in trimer resonance intensity, Liu et

53 H. J. Hoppe, P. N. Barlow, and K. B. Reid, FEBS Lett. 344, 191 (1994). 54 M. H. Li, P. Fan, B. Brodsky, and J. Baum, Biochemisry 32, 7377 (1994). 55 C. G. Long, E. BrasweU, D. Zhu, J. Apigo, J. Baum, and B. Brodsky, Biochemistry 32, 11688 (1993).

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I30]

w e r e able to directly measure by N M R the folding kinetics of the ISNenriched collagenlike triple-helical peptide (Pro-Hyp-Gly)3Ile-Thr-GlyAla-Arg-Gly-Leu-Ala-Gly(Pro-Hyp-Gly)4. Several problems still exist in studying collagen-based peptides. The repetitiveness of the amino acid sequence presents serious problems with resonance overlap. Moreover, because this block copolymer would have the same folded structure, resonance dispersion due to differences in chemical/ magnetic environment is minimal. One way to help overcome this problem is to use selective 15N and/or 13C isotopic enrichment. The problem of strand alignment presents another concern because one relies on an equilibrium among three homostrands to associate the same in all cases. This cannot always be controlled, and various populations with slightly different alignments may arise. Moreover, measurements generally need to be done at low temperature 5 ° or 10 ° in order to shift the equilibrium as far as possible toward a more structured state. A n approach which covalently attaches the three collagen strands to a chemical scaffold 57 appears to be able to circumvent this problem. This novel synthetic approach may also solve the chemical degeneracy problem, as a heterotrimer may be synthesized. A number of N M R conformational analyses also have been performed on collagen-derived telopeptides, that is, nonhelical peptides (20-30 residues) found at the N- and C-terminal ends of collagen. 5s-63 In general, N M R data suggest mostly extended type structures with the presence of some turn populations. For this work, mixed solvents were required for solubility and conformational stabilization. This serves to introduce the next section for discussion.

all. 56

T o w a r d I m p r o v e d C o n f o r m a t i o n a l Stability a n d Better N u c l e a r Magnetic R e s o n a n c e S t r u c t u r e s As should be evident from the above discussion, most peptides in water generally have limited conformational stability, even when the temperature 56X. Liu, D. L. Siegel, P. Fan, B. Brodsky, and J. Baum, Biochemistry 35, 4306 (1996). 57C. G. Fields, C. M. Lovdahl,A. J. Miles, V. L. Matthias Hagen, and G. B. Fields, Biopolymers 33, 1695 (1995). 58X. H. Liu, P. G. Scott, A. Otter, and G. Kotovych,J. Biomol. Strucr Dyn. 8, 63 (1990). 59A. Otter, P. G. Scott, and G. Kotovych,Biochemistry 27, 3560 (1988). 60A. Otter, P. G. Scott, X. H. Liu, and G. Kotovych,J. Biomol. Struct. Dyn. 7, 455 (1989). 61A. Otter, G. Kotovych,and P. G. Scott, Biochemistry 28, 8003 (1989). 62A. Otter, P. G. Scott, and G. Kotovych,Biopolymers 33, 1443 (1993). 63X. Liu, A. Otter, F. G. Scott, J. R. Cann, and G. Kotovych,Z Biomol. Struct. Dyn. 11, 541 (1993).

[301

SOLUTIONNMR ov PEPTIDEFOLDING

663

is lowered. Usually, only short-range NOEs are observed that suggest the presence of turns and/or nascent helix and, as discussed above, few semistable/3-hairpin peptides. Numerous studies, therefore, have been directed toward increasing conformational stability, thereby improving the quantity and quality of N M R observables like J coupling constants and NOEs. Aside from using covalent bonds, that is, -S-S- or peptide cyclization, the four most frequently employed approaches toward improving peptide conformational stability are listed below.

Cosolvents

The most exploited cosolvent known to "stabilize" various conformations in aqueous solution is trifluoroethanol (TFE). Other halogenated aliphatic alcohols, however, such as heptafluorobutyric acid (HFB), may also be used. 64 These solvents generally lower the solution dielectric, help overcome solubility problems, and, more important for this discussion, increase conformational stability. In general, titrations are initially done with the cosolvent in the peptide-water solution in order to optimize percent composition. Usually, cosolvent (i.e., TFE) concentrations used are between 20 and 50%, but this varies from study to study. Other cosolvents may be as active at substantially lower concentrations. 64 The detection of o~ helices in many peptides has been aided by using TFE, which induces formation of helical structure in peptides that are mainly or partly unstructured in aqueous solution but have a helical propensity. 65 The exact mechanism by which T F E stabilizes secondary structure is unknown, but some studies suggest that T F E interacts preferentially with the helical conformation of a peptide to shift the structural equilibrium toward this state. 2122'65,66 It has been shown that the stabilization of helical conformations in protein fragments by T F E tends to be restricted to those regions that are helical in the native protein, although this is not always the case. 67 For the fibrinogen y-chain-derived y27 peptide discussed earlier, N H - N H NOEs are few and weak in the absence of T F E (see Figs. 4 and 5), whereas in the presence of 40% T F E (Fig. 6), NOEs (particularly N H - N H ) are more plentiful and stronger, consistent with the presence of more stable "structure." Helix formation is supported by the presence of helix-constraining i, i + 3 and i, i + 4 NOEs. 68 64y. Yang, S. Barker, M.-J. Chen, and K. H. Mayo, J. Biol. Chem. 268, 9223 (1993). 65F. D. Sonnichsen,J. E. Van Eyk, R. S. Hodges,and B. D. Sykes,Biochemistry 32, 8790(1992). 66A. Jasanoff and A. R. Fersht, Biochemistry 33, 2129 (1994). 67Z. P. Liu, J. Rizo, and L. M. Gierasch, Biochemistry 33, 134 (1994). 68F. Fan and K. H. Mayo, J. Biol. Chem. 270, 24693 (1995).

664

SPECIALIZEDAPPLICATIONS

,-ll

00 o, o 99~~~~

,

'

[30]

I

'

'

'

'

~ 3990 ~39I.

,

,

T

,

I

'

'

'

L4.1

' 8.0

1~~390

~'1

j ~ b

'

8.4

'

~

t

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,

I

I

,

7.9

~

8.4

4o,

,

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I

,

,

,

7.4 PPM

Fro. 6. NH-NH region from a NOESY contour plot. Sample conditionsare the same as in legend for Fig. 3 except for the addition of 40% (v/v) trifluorethanol.

Other examples of TFE-induced stabilization of a-helix conformation include studies on the ribonuclease S-peptide69,7°; peptides derived from bovine pancreatic trypsin inhibitor,71 a peptide 23-mer from the plasma protein transthyretin,72 a peptide fragment from ot-lactalbumin,73 and the 69j. W. Nelson and N. R. Kallenbach,Proteins: Struct. Funct. Genet. 1, 211 (1986). 70j. W. Nelson and N. R. Kallenbach,Biochemistry 28, 5256 (1989). 71j. Kemmink and T. E. Creighton,Biochemistry 34, 12630 (1995). 72j, Jarvis, S. Munro, and D. Craik, Biochemistry 33, 33 (1994). 73LoJ. Smith,A. T. Alexandrescu,M. Pitkeathly,and C. M. Dobson, Structure 2, 703 (1994).

[30]

SOLUTION N M R OF PEPTIDE FOLDING

665

phospholipase A2 fragment residues 38-59. 74 Although helical conformations appear to develop in those regions with a high helical-forming propensity or preexisting helical structure, the stabilizing effects of TFE are not limited to o~-helical peptides. Blanco et al., 75 for example, have observed that TFE also induces nativelike B-hairpin formation in an N-terminal fragment derived from protein G B1 domain. A TFE back-extrapolation procedure 76 has been applied to two barnase-derived synthetic peptides that correspond to sequences 5-21 and 1-36, which contain its first (residues 6-18) and second (26-34) helices. From this analysis, these peptides were found to form 3 and 6% helical structure, respectively, in water at pH 6.3, 25°. 19'76-78 The first helix has been predicted to act as a nucleation site, initiating the folding of barnase 79 and is largely formed in the folding intermediate .80-82 With the use of any cosolvent, caution must be exercised in interpreting data because the solution environment can and often does affect conformational distributions in different ways. For example, the transthyretin peptide 23-mer 72in deuterated dimethyl sulfoxide (DMSO-d6) shows minor populations of turnlike character, whereas nascent helix between residues 5 and 12 was observed in 20% TFE and water.

Micelles/Vesicles

Many peptides have been observed to interact with micelles, vesicles, and membrane bilayers. Using solution NMR techniques, however, resonance broadening due to the long motional correlation times may preclude conformational analysis. In this respect, solid-state NMR techniques82a may be more appropriate to these types of investigations. Nonetheless, in smaller detergent micelles and lipid vesicles, solution NMR has been used success74 M. A. Jimenez, C. Carreno, D. Andreu, F. J. Blanco, J. Herranz, M. Rico, and J. L. Nieto, Biopolymers 34, 647 (1994). 75 F. J. Blanco, M. A. Jimenez, A. Pineda, M. Rico, J. Santoro, and J. L. Nieto, Biochemistry 33, 600 (1994). 76j. Sancho, J. L. Neira, and A. R. Fersht, J. Mol. Biol. 224, 749 (1992). 77j. Sancho, L. Serrano, and A. R. Fersht, Biochemistry 31, 2253 (1992). 78 A. D. Kippen, V. L. Arcus, and A. R. Fersht, Biochemistry 33, 10013 (1994). 79 L. Serrano, J. T. Jr. Kellis, P. Cann, A. Matouschek, and A. R. Fersht, J. Mol. Biol. 224, 783 (1992). 80 A. Matouschek, J. T. Kellis, Jr., L. Serrano, M. Bycroft, and A. R. Fersht, Nature (London) 346, 440 (1990). 81 A. Matouschek, L. Serrano, and A. R. Fersht, J. Mol. Biol. 224, 819 (1992). 82 A. R. Fersht, FEBS Lett. 325, 5 (1993). s2a T. A. Cross, Methods Enzymol. 289, [31], 1997 (this volume).

666

SPECIALIZED APPLICATIONS

[30]

fully. Chupin et al., s3 for example, have studied the PhoE signal peptide 21mer in a micellar environment and showed that it formed N- and C-terminal a-helical conformations which were separated by a kink at a central glycine. Depending on the size of the macromolecular aggregate and the lifetime of the "bound" peptide, use of the transferred NOE experiment to derive structural information may be appropriate; this is discussed later in this article. P e p t i d e - Peptide Interactions

In protein folding in general conformational units associate to form a more stable fold. This idea has been carried through to peptide conformational studies by mixing in solution two peptides that would normally be brought together in the native protein. Wu et a1.,84for example, investigated a peptide model for an early cytochrome c folding intermediate consisting of a noncovalent complex between a heme-containing N-terminal fragment (residues 1-38) and a synthetic peptide corresponding to the C-terminal helix (residues 87-104). Those studies indicated that a partially folded intermediate with interacting N- and C-terminal helices is formed at an early stage of folding when most of the chain is still disordered. NMR and CD indicate that the isolated peptides are largely disordered, but, when combined, they form a flexible, yet tightly bound complex with enhanced helical structure. These results emphasize the importance of interactions between marginally stable elements of secondary structure in forming tertiary subdomains in protein folding. Self-association of peptides also clearly can stabilize conformational populations. For example, the 33-amino acid/3-sheet peptide derived from PF4 (residues 23-55) sl at low concentration exists in aqueous solution in a "random coil" distribution of highly flexible conformational states. Some preferred conformation, however, is observed, particularly within a relatively stable chain reversal from Lys-45 to Arg-49. As the peptide 33mer concentration and/or temperature is increased, nativelike antiparallel /3-sheet folding results. Sheet formation is thermodynamically linked with tetramerization of the peptide, which occurs on the slow chemical shift exchange time scale. Other/3-sheet-forming peptides also have been recognized to self-associate and exhibit considerably more stable conformations.8 Terzi et al. 8s had similar findings with the aggregation of fragment 25-35

83 W. Chupin, J. A. Killian, J. Breg, H. H. de Jongh, R. Boelens, R. Kaptein, and B. Kruijff, Biochemistry 34, 11617 (1995). 84 L. Wu, P. Laub, G. Elove, J. Carey, and H. Roder, Biochemistry 32, 10271 (1993). 8s E. Terzi, G. Holzemann, and J. Seelig, Biochemistry 33, 1345 (1994).

[30]

SOLUTION N M R o v PEPTIDE FOLDING

667

of the/3-amyloid protein [/3AP(25-35)OH] studied under a variety of conditions. D e N o v o Design A p p r o a c h

Most of what has been presented above concerns peptides with sequences derived from native proteins. An alternative approach aimed at improving conformational stability uses basic folding principles to design peptides de novo. A number of these de n o v o designed peptides have been designed to promote o~-helical and/or turn properties. Regan and DeGrado 86 designed a relatively stable helical peptide. Making use of the hydrophobic effect to engineer a stable helix fold, Hecht et al. 87 was one of the first to design a four-helix bundle. Fairman et aL 88 designed and characterized another four-chain coiled coil and found that increasing the monomer peptide length in the helix from 21 to 28 to 35 residues resulted in tetramer stabilities in terms of peptide monomer concentration that ranged from 0.18 mM to 51 nM to 0.28 pM, respectively. Munoz et aL 37 have identified a recurrent, stabilizing local structural motif found at the N termini of c~helices that they refer to as the hydrophobic-staple; a more stable helix-turn structure is formed when hydrophobic residues from the N-terminal side of a turn interact with hydrophobic residues from the helix. On the basis of such hydrophobic stabilization effects, others have designed various a/o~ and od/3 peptides. 89-91 Whereas most of these peptides form marginally stable helix structures at best, Struthers et aL 91 have designed an odfl peptide 23-mer based on a zinc finger motif, which in the absence of coordinating zinc or disulfide bonds forms a compact structure with an N-terminal amphipathic c~-helix folded onto a two-stranded amphipathic/3 sheet. Although de n o v o design of o~helix, helix bundle, and od/3 peptides has been successful, designing water-soluble, purely /3-sheet-containing peptides has met with limited success. Designing/3-sheet peptides has proved more complicated primarily because of their limited solubility via aggregation in water and because of the nature of/3-sheet folding, which is dictated by long-range interactions. A small peptide 12-mer, however, has been designed which is water soluble, remains monomeric, and does form some 86 L. Regan and W. DeGrado, Science 241, 976 (1988). 87 M. Hecht, J. Richardson, D. Richardson, and R. Ogden, Science 249, 884 (1990). 88 R. Fairman, H. G. Chao, L. Mueller, T. B. Lavoie, L. Shen, J. Novotny, and G. R. Matsueda, Protein Sci. 4, 1457 (1995). 89 y. Fezoui, D. L. Weaver, and J. J. Osterhout, Protein Sci. 4, 286 (1995). 90 D. J. Butcher, M. D. Bruch, and G. R. Moe, Biopolymers 36, 109 (1995). 91 M. D. Struthers, R. P. Cheng, and B. Imperiali, Science 271, 342 (1996).

668

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[301

highly transient B-hairpin-like structure. 92 The beta-bellin s e r i e s 93 and beta-doublet 9~ peptides exemplify initial attempts at designing larger /3-sheet sandwich peptides; these peptides, usually about 30 residues in length, show limited solubility in water primarily at lower p H values and n o n c o m p a c t fl-sheet conformational properties as monitored by C D and N M R . Owing t o limited water solubility of beta-bellin 12, for example, N M R assignments could be m a d e only in dimethyl sulfoxide, and no longrange N O E s were observed, indicating the absence of stable folded structure. 93 B e t a - D o u b l e t 94 which has the same predicted antiparallel/3-sheet motif as beta-bellin, was water-soluble, albeit at low pH; gave a typical /3-sheet CD trace, but failed to show any compact folding as monitored by N M R , that is, it gave a 1H N M R " r a n d o m coil" spectrum. 7 Beta-Bellin I 4 D 95 is the best in that series and shows good solubility at millimolar concentrations up to p H 5.5. However, it does not give a good compact B-sheet fold even with a covalent disulfide bond between two sandwiched monomers. O t h e r amphipathic/3-sheet forming peptides have b e e n derived from proteins in the ot-chemokine family. Studies on/3-sheet domain peptides derived from IL-8, 8 Gro-t~ (growth-related protein-a), s and PF4, 5I as well as those on beta-bellins 93,95 and beta-doublet, 94 led to some general guidelines for designing water-soluble, self-association-induced B-sheet-forming peptides, s N M R conformational analyses show that formation of/3-sheet structure is thermodynamically linked to p e p t i d e - p e p t i d e associations as discussed in the previous section. O n e of these B-sheet sandwich peptide 33-mers was found to fold compactly as a t e t r a m e r c o m p o s e d of two heterodimers. 96 Although the three-stranded antiparallel/3-sheet folds in monom e r subunits were generally the same, two types of dimers f o r m e d by continuing m o n o m e r i c / 3 sheets into a six-stranded sheet, each with differently aligned interracial antiparallel/3 strands. H e t e r o d i m e r s then associated by sandwiching their amphipathic hydrophobic surfaces. In the future, it is expected that d e n o v o designs of various desired peptide conformations will continue with the use of n o n c o m m o n 9I and D - i s o m e r 97 amino acids. 92V. Sieber and G. R. Moe, Biochemistry 35, 181 (1996). 93j. S. Richardson, D. C. Richardson, N. B. Tweedy, K. M. Gernert, T. P. Quinn, M. H. Hecht, B. W. Erickson, Y. Yang, R. D. McClain, M. E. Donlan, and M. C. Surles, Biophys. J. 63, 1185 (1992). 94T. P. Quinn, N. B. Tweedy, R. W. Williams, J. S. Richardson, and D. C. Richardson, Proc. Natl. Acad. Sci. U.S.A. 91, 8747 (1994). 95y. Yah and B. W. Erickson, Protein Sci. 3, 1069 (1994). 96E. Ilyina, V. Roongta, and K. H. Mayo, Biochemistry 36, 5245 (1997). 97V. Bobde, Y. U. Sasidhar, and S. Durani, Int. J. Pept. Protein Res. 43, 209 (1994).

[30]

SOLUTIONNMR OF PEPTIDEFOLDING

669

Transferred Nuclear Overhauser Effect: Peptide Ligand-Receptor Interactions Since the late 1980s, use of the transferred NOE (TRNOE) has made possible conformational studies of small peptides in the "receptor" bound state. The basic experiment employed is the usual NOESY. What differentiates a TRNOE experiment from a standard NOE experiment is the presence of "ligand" and "receptor" components in the experimental system. Figure 7 depicts the basic situation for the TRNOESY experiment. In the presence of an appropriate receptor, a smaller peptide ligand can exist either free in solution or bound to its receptor. This "receptor" could be an enzyme, a membrane receptor, plasma protein, etc. Although the "receptor" molecular mass could be anything greater than about 25,000 Da, a molecular mass of 200,000 Da will be used for the purpose of this discussion. A peptide of about 12 residues (1400 Da) is often used. In solution, the larger receptor is considerably less mobile than the peptide. Overall tumbling correlation times, ZR and TF, are approximated to be i × 10-7 and i × 10 -9 sec, respectively, and the correlation time for the complex is about that of the receptor itself. Therefore, the effective correlation time, zef~, in terms of the peptide ligand, is the population-weighted average of the bound and free ligand states: (1)

"/'eft = XBTB -[- XFTF

where X is the mole fraction of ligand in the bound (B) and free (F) states. In this example, 1% peptide bound will contribute nearly equally to 0"ereas 99% free. For this reason, usually 5% or less of bound ligand is experimentally required, Furthermore, zef~is optimized by adjusting the mole fraction

Receptor

Peptide ligand

~R

~F

Ligand bound receptor

~B

FIG.7. Schematicrepresentationof a peptide ligandinteractingwith a "receptor."

670

SPECIALIZEDAPPLICATIONS

1301

of bound and free ligand such that observed ligand resonances for the free state dominate the spectrum; those of the receptor are often either too broad to be observed or can be filtered experimentally. The N O E can be expressed in the following proportionality as: N O E oc f(reff)/r 6.

(2)

where rq is the interproton distance. The numerator, f('reff), indicates that the N O E is a function of Tell. From Eq. (2) it is evident that increasing zeff with some fraction of the peptide bound to receptor can significantly increase the N O E magnitude. Moreover, this experiment is most easily interpreted when the N O E for the free ligand is either positive or near zero, because in the large receptor complex the N O E will be negative; corrections for NOEs arising from the larger free state population are thereby obviated. In this case, the absence of free ligand (peptide) NOEs becomes an advantage. One further consideration is the interplay of the dipolar cross-relaxation rate, which dictates how fast the NOE will build up and the lifetime of ligand-receptor complex, that is, receptor off-rate, koff. In the receptorbound state, NOEs build up considerably faster than in the ligand-free state, primarily owing to the much larger value of ZB. If the ligand remains on the receptor for a time longer than the inverse of the N O E buildup rate, various indirect cross-relaxation processes would occur and no T R N O E s would be observed. The key to adjusting experimental parameters for an effective T R N O E lies in making the ligand off-rate from the receptor complex fast relative to the N O E buildup or cross-relaxation rate. In this case, bound-state NOEs develop in the receptor complex and the ligand is rapidly released; in the ligand-free state, relaxation processes occur much more slowly, and ligand NOEs which developed in the bound state remain for a time and can be detected in the usual NOESY experiment. In general, the equilibrium dissociation constant should be no smaller than about l0 X 10 -6 M. With a diffusion-limited ligand on-rate of 5 x 10 7 M 1 sec-1, the ligand-receptor off-rate would be 500 sec -I. For a 200,000-Da receptor complex and an interproton distance of 2.5 ~ , the cross-relaxation rate would be about 25 sec 1. Another useful observable in this experiment is the chemical shift, which also presents itself as a weighted average of free and bound ligand states: ~obs : X B ~ -I- XF(~F.

(3)

By analyzing chemical shift changes between free and bound ligand or by varying the ratio of free to bound ligand, it is sometimes possible to argue that resonances most shifted (and broadened) are more directly involved in receptor binding.

[301

SOLUTIONNMR OF PEPTIDEFOLDING

671

This brief overview merely considers some of the salient aspects of the TRNOE experiment. A more thorough analysis of TRNOE data involves, for example, considering various internal motions and implementing a full relaxation matrix approach in which a chemically exchanging multispin system is analyzed. More explicit details of the theory behind the TRNOE can be found in several reviews. 98-1°° Use of the TRNOE to investigate receptor-bound peptide conformation can be exemplified by several 2D NMR studies where "receptor" takes on the role of enzymes: thrombin, l°1-1°3 phosphoglycerate kinase, TM cyclophilin, 1°5 the plasma protein fibrinogen,m6 muscle proteins, ~°7 micelles,3° lipid vesicles, ~°8 and a membrane glycoprotein, integrin GPIIb/IIIaJ °9 Most of these TRNOE studies were done using synthetic peptides less than 20 residues in length. With thrombin as the "receptor," for example, Ni et aL lm found that TRNOEs of the 14-residue C-terminal fragment (residues 52-65) of hirudin (a 65-residue protein) are consistent with an a-helical structure involving residues 61 to 65 and a hydrophobic cluster involving Phe-56, Ile-59, Try-63 and Leu-64, with all charged residues lying on the opposite face. Thrombin-induced resonance broadening of peptide side chains 55 to 65 argues that this sequence is the predominant binding site of hirudin fragments with thrombin.

Conclusion Much has been learned concerning basic principles of protein folding from studies on synthetic peptides derived from native protein sequences. This information has been almost immediately incorporated into the de n o v o design of novel peptides with a desired fold. As the state of the art

98 R. E. London, M. E. Perlman, and D. G. Davis, J. Magn. Reson. 97, 79 (1992). 99 m. P. Campbell and B. D. Sykes, Annu. Rev. Biophys. Biomol. Struct. 22, 99 (1993). 10oF. Ni, Prog. NMR Spectrosc. 26, 517 (1994). 101 F. Ni, Y. Konishi, and H. A. Scheraga, Biochemistry 29, 4479 (1990). 102 F. Ni, D. R. Ripoll, and E. O. Purisima, Biochemistry 31, 2545 (1992). 103 j. Srinivasan, S. Hu, R. Hrabal, Y. Zhu, E. A. Komives, and F. Ni, Biochemistry 33, 13553 (1994). 104 M. Andrieux, E. Leroy, E. Guittet, M. Ritco-Vonsovici, B. Mouratou, P. Minard, M. Desmadril, and J. M. Yon, Biochemistry 34, 842 (1995). lO5L. T. Kakalis and I. Armitage, Biochemistry 33, 1495 (1994). lo6 L. J. Yao and K. H. Mayo, Biochem. J. 315, 161 (1996). 107A. P. Campbell and B. D. Sykes, J. Mol. BioL 222, 405 (1991). 1~ Z. Wang, J. D. Jones, J. Rizo, and L. M. Gierasch, Biochemistry 32, 13991 (1993). lo9 K. H. Mayo, F. Fan, M. P. Beavers, A. Eckardt, P. Keane, W. J. Hoekstra, and P. AndradeGordon, Biochemistry 35, 4434 (1996).

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[3 II

improves, more compactly folded structures are being made. NMR is perhaps the only technique which can provide such detailed information regarding peptide conformational distributions and preferred structures in solution.

[31] S o l i d - S t a t e N u c l e a r M a g n e t i c R e s o n a n c e Characterization of Gramicidin Channel Structure

By T. A. CRoss Introduction Solid-state nuclear magnetic resonance (NMR) is a powerful technique for studying molecular structure in complex environments. Lipid bilayers represent such a complex environment. Two-dimensional or three-dimensional crystallization of membrane proteins in bilayer environments is possible but very challenging. 1,2 Similarly the study of membrane proteins by solution NMR is substantially complicated by the presence of lysolipids or detergents, thereby limiting this technique to very small molecular weight proteins. Consequently, there have been relatively few membrane protein structures solved. Similarly, the structure of fibrous proteins and large protein-protein or protein-nucleic acid complexes have been rarely solved even at modest resolution. Solid-state NMR neither requires crystallization nor isotropic environments; in fact, the more rigid, the better. 3'4 Hence this spectroscopic approach has a great deal of potential for studies of these proteins. This article describes the current state of the development of solid-state NMR structural methods for these environments with special attention to the first high resolution structure of a membrane-bound polypeptide by solid-state NMR, gramicidin A . 5"6 Unlike an aqueous environment, the lipid environment is heterogeneous as a result of the low dielectric lipid bilayer interstices, the bulk aqueous environment, and the considerable interface region. 7 Furthermore, lipids 1T. Tsukihara, H. A o y a m a , E. Yamashita, T. Tomizaki, H. Yamaguchi, K. Shinzwa-Itoh, R. Nakashima, R. Yaono, and S. Yoshikawa, Science 272, 1136 (1996). 2 W. Kohlbrandt, Q. Rev. Biophys. 25, 1 (1992). 3 S. J. Opella, Methods Enzymol. 131, 327 (1986). 4 T. A. Cross and S. J. Opella, Curr. Opin. Struct. Biol. 4, 574 (1994). 5 R. R. Ketchem, K.-C. Lee, S. Huo, and T. A. Cross, J. Biomol. N M R 8, 1 (1996). 6 R. R. Ketchem, B. Roux, and T. A. Cross, submitted. 7 S. H. W h i t e and W. C. Wimbley, Curr. Opin. Struct. Biol. 4, 79 (1994).

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