Protein and peptide secondary structure and conformational determination with vibrational circular dichroism

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Protein and peptide secondary structure and conformational determination with vibrational circular dichroism Timothy A Keiderling Vibrational circular dichroism (VCD) provides alternative views of protein and peptide conformation with advantages over electronic (UV) CD (ECD) or IR spectroscopy. VCD is sensitive to short-range order, allowing it to discriminate β-sheet and various helices as well as disordered structure. Quantitative secondary structure analyses use protein VCD bandshapes, but are best combined with ECD and IR for balance. Much recent work has focused on empirical and theoretical VCD analyses of peptides, with detailed prediction of helix, sheet and hairpin spectra and site-specific application of isotopic substitution for structure and folding. Addresses Department of Chemistry, University of Illinois at Chicago, 845 West Taylor Street, Chicago, IL 60607-7061, USA; e-mail: [email protected] Current Opinion in Chemical Biology 2002, 6:682–688 1367-5931/02/$ — see front matter © 2002 Elsevier Science Ltd. All rights reserved. Published online 30 August 2002 Abbreviations Aib amino-isobutyric acid ECD electronic circular dichroism FTIR Fourier transform infrared PLL poly-L-lysine PLP II poly-L-proline II ROA Raman optical activity VCD vibrational circular dichroism

Introduction As the focus of developing research in the broad arena of Chemical Biology moves from genomics to proteomics, the ability to obtain structural information about protein and proteinic systems on a relatively rapid time scale will be an increasingly important factor in moving from sequence (or genetics) toward function (proteomics, which is intimately tied to the protein fold). Optical spectroscopy and circular dichroism of electronic transitions (ECD) in the UV have long been important tools for determination of secondary structure and monitoring of structural change [1,2]. However, the information content of UV absorption spectra is limited by the inherently low resolution, high overlap and conformational insensitivity of the few accessible electronic transitions. The differentially polarized absorption of ECD adds chiral sensitivity, and extension of such measurements to the vacuum UV (e.g. with synchrotron radiation) significantly enhances information content [3,4]. By contrast, in the vibrational region of the spectrum, characteristic transitions of the amide functionality are naturally well-resolved from most other transitions, such as side-chain modes. Spectral separation of structurally

characteristic vibrational modes (or chromophores) underlies IR and Raman spectroscopy, in which structure is correlated to frequencies, whereas in ECD it is to bandshapes enhanced by sign variation. The desire to access multiple localized transitions with conformationally sensitive bandshapes led to the development of vibrational CD (VCD) and its differential scattering analogue, Raman optical activity (ROA) [5,6•,7]. VCD originally centered on smallmolecule conformational analysis and theory, and still has important roles there, particularly for chiral drug enantiomeric determination studies [8,9•,10]. Applicability of VCD (and ROA) to studies of biopolymers (peptides, nucleic acids, carbohydrates) developed later [6•,7,11]. Because of space considerations, this review focuses only on recent developments in protein and peptide applications of VCD for secondary structure analysis. Numerous reviews of ECD, IR and ROA applications are available [2,7,12]. The local character of vibrational excitations gives VCD some important properties. Overlap with aromatic modes (a major problem for ECD) is basically resolved in the IR and becomes even less of an interference for VCD because side chains are locally achiral and do not couple strongly to the amides. Furthermore, vibrational coupling tends to be short range, primarily to the next residue (or to a hydrogenbonded one), and the dipole moments are relatively weak, so VCD samples structure more locally than does ECD [6•,11,13]. Secondary structure information can be derived from amide VCD bandshapes, which reflect the helicity of the backbone, much like ECD. Frequencies are also useful (especially relative to the IR) but are perturbed by solvent and environment, whereas the bandshape is less affected. Analyses of this structural information content from VCD spectra for biological systems have ranged from totally empirical correlation to fully theoretical predictions. For proteins, a statistically based, empirical VCD secondary structure prediction model has been developed, paralleling ECD and IR [14] methods. While for peptides, ab initio quantum mechanical VCD simulations are possible [6•,15••].

Instrumentation Most VCD measurements have been made with instruments constructed by modifying either a dispersive or Fourier transform IR (FTIR)-based absorption spectrometer. Commercial FTIR-based VCD instruments are now available [16–19,20•] and provide high quality spectra for small molecules in non-aqueous solutions. Aqueous biopolymer VCD imposes high demands, particularly with regard to baseline artifacts. Consequently, stable high-throughput dispersive instruments continue to provide excellent

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Figure 1 Comparison of typical amide I′ (N-deuterated, left), amide I & II (middle) and amide III (right) VCD spectra for proteins in solution that have different dominant secondary structures. Spectra are for hemoglobin (HEM, top, highly helical); concanavalin A (CAN, highly β-sheet, no helix); ribonuclease A (RNA, second from bottom, sheet and helix mixed); and casein (CAS, bottom, a ‘random coil’ protein with no extensive secondary structure). Amide I′ (D2O) [14] and amide I+II (H2O) [27] VCD are normalized to A = 1 for the amide I (I′), but the amide III VCD (H2O) have an independent normalization [26].

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biopolymer VCD data [6•,21,22]. A potential breakthrough using two modulators and two lock-in amplifiers was recently reported to cancel out birefringence effects and enhance VCD signal-to-noise ratio [20•]. An alternate approach, using digital signal processing of the timedependent modulated signal in a step-scan FTIR-based experiment, allows elimination of lock-ins and simultaneous detection of multiple modulations [19]. Efforts to use a polarizing beam splitter for polarization modulation in the interferometer have promise but, as yet, have not found practical VCD application [23].

Protein applications Proteins with different folds give characteristic VCD shapes, which vary most for the amide I mode (C=O stretch), but are also easily detectable for the broader amide II and III (very weak) modes (N–H deformation and C–N stretch). Examples are shown in Figure 1. Surveys of protein (and even nucleic acid) spectra show that secondary structure determines the dominant contributions to the VCD shape [6•,11], although results for ligand bands in heme proteins probably indicate tertiary interactions [24]. This bandshape dependence led to development of factor-analysis-based schemes to separate spectral components and develop correlations between their contributions (loadings) and the fractional secondary structure (e.g. % helix, % sheet, etc.) [14]. These relate

to principle component analysis methods for protein ECD [1,2,25]. Analyses of amide I′ (proteins in D2O, N–D exchanged), plus amide II, III, and amide I+II (proteins in water) VCD and combinations of VCD and ECD or FTIR data all yield secondary structure at some level [14,26–28]. The important aspects were that VCD sensed sheet and other structural elements (including turns) differently than did ECD, which in turn was superior for helix determination. Combining them gave better determination of all components, especially minimizing the impact of outliers on the prediction scheme [11,14,27]. Detailed tests of predictive ability of the dependence of factor analysis loadings on fractional secondary structure, using a restricted multiple regression method, showed that only a few loadings were needed to obtain a statistically reliable structure correlation but these varied for different structural components [14,27]. More recently, H/D exchange-correlated FTIR spectra were shown to provide even better analyses, suggesting such an approach to VCD analysis might be beneficial [28]. IR, VCD, ECD and Raman spectral features have also been related to each other using 2-D correlation analysis based on helix and sheet content [29,30]. Similar methods have been used to assign the order of secondary structure

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changes in avidin and avidin-biotin complexes as a function of temperature [31]. Determination of the number of uniform segments (or average lengths of helices and sheet strands) is possible with VCD, FTIR and ECD spectra [32,33•]. The mechanism of thermal unfolding of ribonuclease T1 has been proposed to involve loss of the single helical segment and shortening of sheet segments based on such data [34]. An alternative approach is to study stability and folding of isolated segments of the protein sequence and use that to understand the protein, as was done for the human chorionic gonadotropin β subunit [35•].

Other conformational discriminations have been attempted with VCD, but are less well-established. Despite early optimism, evaluation of the effect of twisting the sheet indicates difficulty in discriminating antiparallel and parallel β-sheets [47••]. Antiparallel pairs of β-strands coupled by a type I′ or II′ turn (especially if stabilized with a D-Pro-Gly linkage [48–50]) do give characteristic spectra in non-aqueous environments [51••]. These characteristics are still seen, though broadened, in aqueous solution, implying that the strands are more frayed [52]. Gramicidin D spectra in SDS micelles and lipid vesicles have been proposed to model β6.3 helical VCD [17].

Peptide applications Empirical correlations of peptide VCD bandshapes with secondary structure were developed in the 1980s, particularly for polypeptides in non-aqueous environments [6•,36,37•]. The key element was determination that the amide VCD depended on the conformation of the peptide chain, and was independent of other functional groups. The helical chirality was the major bandshape criterion, a point demonstrated by VCD of poly-γ-(α-phenethyl)-L-glutamates having side chains of different chirality (R and S) but developing the same sign amide I and II VCD [38]. Similarly, (LysAla2)n oligopeptides yield amide VCD independent of interacting porphyrins [39], reflecting earlier studies that showed peptides with aromatic residues maintain established patterns [40,41]. For systems of uniform structure, higher levels of structural definition were possible. A major success was discrimination between 310- and α-helices, which, because of their common right-hand helical structures, tend to give rise to very similar spectral bandshapes [42,43]. Model 310-helices can be stabilized for sequences containing a high fraction of αMe-substituted residues, such as Aib (amino-isobutyric acid). VCD spectra of mixed Aib–Ala sequences coupled to theoretical modeling showed these VCD bandshapes are qualitatively independent of αMe effects [44•]. VCD can distinguish 310- and α-helical structures when either is dominant and well-defined, but, as often happens in proteins, if the 310- component is a minor fraction compared with the α-helical component, quantitative determination of their separate contributions is unlikely. Another significant contribution of peptide VCD to conformational studies was confirmation of an old proposal [45] that the random coil conformation, such as seen in poly-L-lysine (PLL) or poly-L-glutamic acid at neutral pH contained a significant component of ‘extended helix’ with a left-handed twist. Length-dependent studies of PLL and proline oligomers showed that all gave rise to a characteristic poly-L-proline II (PLP II) VCD spectrum that broadened and weakened for short or higher temperature PLL samples [13,37•,46]. VCD sensed significant structure formation for Pron oligomers (n > 4) with characteristic PLP II spectra. The random coil contains locally left-hand twisted strands whose amide carbonyls project into solution, lending stability in water.

Sensitivity to local structure suggests that VCD should develop characteristic patterns for β-turns and other non-repeating structures. Cyclo(Cys-Pro-Xxx-Cys), with an S–S-linked 14-member ring, can form type I and II turns depending on the Xxx residue [53], but the VCD vary considerably depending on the solvent properties. Turn residues in proteins also interact more with the environment being on the surface of a fold. Cyclic(Pro–Gly)3 peptides have shown a sharp dependence on metal complexation, implying a conformational variation [54]. VCD of cyclo[Gly-Pro-Gly-Ala-Pro], which forms γ- and β-turns has been the subject of theoretical spectral modeling [55]. Similarly, just the Xxx-D-Pro-Gly-Xxx-stabilized β-turn yields a strong VCD signal that is modeled well by ab initio VCD simulations [56]. Turns are junctions between different structural types, which contribute to unfolding mechanisms. VCD studies of alanine-rich peptides showed sensitivity to an intermediate state in which helical residues are coupled to coil segments via a junction whose formation in the transition was sensed by VCD and FTIR. Its temperature dependence was characterized by factor analysis of the bandshapes [57]. The ensemble of intermediate structures had a helix-coil junction whose population grew and declined with increasing temperature. While the junction may have 310-helical character, their VCD spectra confirmed these Ala-peptide conformations to be predominantly α-helical. A major limitation of optical spectroscopy is lack of site-specific resolution in that vibrational transitions corresponding to the same sort of motion on different repeating residues will spectrally overlap. However, because these transitions result from nuclear motion, an added variant is available for IR and VCD of peptides. Isotopic substitution of specific residues can be used to shift transitions on those sites to spectroscopically unique positions, yet maintain interpretable bandshapes yielding site-specific conformational information. Model α-helical peptides labeled with 13C on the amide C=O have an amide I component shifted ~40 cm–1 to lower frequency [58], and if labeled with 13C and 18O the shift is ~65 cm–1 [59]. VCD studies of Ac-AAAA(KAAAA)3Y-NH2 at ~5°C have shown that four residues in sequence also yield a VCD bandshape characteristic of an α-helix unless they are at the C-terminus [60••], as shown in Figure 2. This

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Experimental (left, low temperature, 5°C) and theoretical (right, α-helical conformation) amide I′ VCD (top) and IR (bottom) spectra for Ac-AAAA(KAAAA)3-Y-NH2 in D2O. Comparison of effects of 13C substitution on the amide C=O, for unsubstituted (dotted line), N-terminal substituted (solid line) and C-terminal substituted (dashed dot line) peptides with four labeled Ala in succession. The match of the N-terminal results shows the α-helical structure is maintained but the mismatch with the C-terminal results shows it to be unwound.

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work demonstrated that the termini were less stable than the central residues and that the peptide unfolds from the ends, which is consistent with NMR and EPR results [61,62]. Even just two adjacent labeled residues yield detectable α-helical signals although they are more difficult to detect [15••].

Theoretical simulations The major recent development for peptide VCD is the ability to simulate spectra for various conformations based on ab initio quantum mechanical force fields and the corresponding intensity parameters, termed atomic polar and axial tensors, as developed with the magnetic field perturbation theory of Stephens [9•]. Although such fundamental studies ordinarily are not practical for biological problems, simpler theoretical interpretive models of peptide VCD are unreliable. Because only in ideal cases can well-defined conformations be confidently assigned to small peptides, a reliable theoretical method can be used to explore conformational sensitivity of spectra since all conformations are computationally accessible. Initial studies in this field focused on glycine-based peptides

containing two coupled amide functions but constrained to conventional secondary structure geometries [63]. Subsequently, larger and even solvated small peptides have been modeled [15••,44•,47••,60••,64,65,66•]. Although peptides of biological interest and proteins are typically too large for such ab initio calculations with current computer capabilities, Bour et al. [67] have developed a method for transfer of force field and intensity parameters from a smaller peptide to a larger one that shares its conformation. IR and VCD spectra for systems containing as many as 65 residues have been stimulated in this manner [68]. An alternative method using neural networks to correlate computed vibrational spectra with conformation for various structures has recently been reported [69]. The important issue of solvent effects is also beginning to be addressed theoretically for peptide spectra [66•,70,71]. Initial studies focused on helices and used tripeptides for transfer to larger oligomers [64], but better results were obtained by using oligomers containing seven or five amides to model α and 310-helices, respectively [15••]. Although the spectra are very similar to those simulated

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with tripeptides, with this larger-sized ‘small’ peptide the central residue is fully hydrogen bonded and thus better coupled to other residues. Accurate simulations of α-helical and left-handed 31-helical (PLP II-like) amide I and II absorption and VCD were obtained (Figure 2) [15••,60••]. The α-helical low-energy weak negative VCD amide I′ component depended on length [56], but the 31-helical result did not, probably because of the lack of internal hydrogen bonds. The qualitative difference between α- and 310-helical VCD and even the dependence on composition (Aib vs. Ala) could be simulated [44•]. That such subtle effects, due to the electronic structure effects of α-methylation, can be modeled points to the simulation’s accuracy. Simulations of thermal denaturation of α-helical peptides with various isotope substitutions [60••] showed the C-terminal region to be unwound (not α-helical) and confirmed the utility of modeling the random-coil, thermally denatured state as an extended helix [37•]. At minimum, two sequential isotope substitutions are needed to develop a helical VCD, and separation of the labels by an unlabeled residue weakens the 13C VCD and distorts its sign pattern. Parallel and anti-parallel β-sheet conformations were the subject of another theoretical study, which indicated similar weak predominately negative VCD when both were twisted [47••]. On the other hand, extended, flat (long multi-stranded) antiparallel β-sheet structures have unique IR and VCD patterns, with an intense lowfrequency component (IR), reflecting that often found for model polypeptide β-sheets such as high pH PLL and for aggregated peptides and proteins. This unique structure also has a position-sensitive, intense 13C pattern when isotope substituted on alternate residues [68] which acts as a site-specific amplification effect and suggests a potential for detecting local, initial anti-parallel sheet formation. Extending these methods to hairpins required a modified transfer method to encompass multiple geometries. Predicted amide I spectra were in qualitative agreement with VCD for model hairpins based on D-Pro-Gly type I′ and II′ turns [51••,52,56].

Conclusions A number of useful applications for VCD in biopolymer conformational studies have been established. With commercialization of instrumentation, these will surely increase. Presently, the major impact is on peptide studies where conformation can be partially controlled by external perturbations and where theory provides an external test of the analysis. Protein applications will increase, especially as signal-to-noise ratio is improved and applications become feasible for equilibrium folding studies, in which local structural changes are important. VCD can complement IR, Raman, ROA and ECD analyses of complex structural changes, and these all sense biopolymer structure in solution on a rapid time scale appropriate to biological activity. Although all have limitations, together

they support and correct interpretations derived from each independently.

Acknowledgement This work was recently supported in part by grants from the Petroleum Research Fund administered by the American Chemical Society and by the Research Corporation.

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• of special interest •• of outstanding interest 1.

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Keiderling TA: Peptide and protein conformational studies with vibrational circular dichroism and related spectroscopies. In Circular Dichroism: Principles and Applications, edn 2. Edited by Berova N, Nakanishi K and Woody RA. New York: Wiley-VCH; 2000:621-666. This review encompasses peptide and protein conformational applications and methods in detail, including dispersive VCD techniques. Examples of empirical and theoretical peptide applications, including isotopes, and statistical analyses of protein VCD are discussed at length, including the basic methods employed. 7.

Barron LD: Hecht L: Vibrational Raman optical activity: from fundamentals to biochemical applications. In Circular Dichroism, Principles and Applications, edn 2. Edited by Nakanishi K, Berova N and Woody RW. New York: Wiley-VCH; 2000:667-701.

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Stephens PJ, Devlin FJ, Amouche A: Determination of the structures of chiral molecules using vibrational circular dichroism. In Chirality: Physical Chemistry. ACS Symposium Series, vol 810. Edited by Hicks JM. New York: Oxford University Press; 2002:18-33. The authors present a compact summary of their density functional theory/GIAO method of computing VCD spectra with the magnetic field perturbation theory and apply it to the simulation of spectra for molecules ranging from methyl oxirane (a small, rigid test case) to large species such as Trogler’s base and flexible cases containing rotational isomers. 10. Stephens PJ, Devlin FJ: Determination of the structure of chiral molecules using ab initio vibrational circular dichroism spectroscopy. Chirality 2000, 12:172-179. 11. Keiderling TA: Vibrational circular dichroism applications to conformational analysis of biomolecules. In Circular Dichroism and the Conformational Analysis of Biomolecules. Edited by Fasman GD. New York: Plenum; 1996:555-598. 12. Mantsch HH: Chapman D: Infrared Spectroscopy of Biomolecules. Chichester, UK: Wiley-Liss;1996. 13. Dukor RK, Keiderling TA: Reassessment of the random coil conformation: vibrational CD study of proline oligopeptides and related polypeptides. Biopolymers 1991, 31:1747-1761. 14. Pancoska P, Bitto E, Janota V, Urbanova M, Gupta VP, Keiderling TA: Comparison of and limits of accuracy for statistical analyses of

Secondary structure and conformational determination with VCD Keiderling

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Baumruk V, Pancoska P, Keiderling TA: Predictions of secondary structure using statistical analyses of electronic and vibrational circular dichroism and Fourier transform infrared spectra of proteins in H2O. J Mol Biol 1996, 259:774-791.

28. Baello B, Pancoska P, Keiderling TA: Enhanced prediction accuracy of protein secondary structure using hydrogen exchange Fourier transform infrared spectroscopy. Anal Biochem 2000, 280:46-57. 29. Pancoska P, Kubelka J, Keiderling TA: Novel use of a static modification of two-dimensional correlation analysis. Part I: comparsion of the secondary structure sensitivity of electronic circular dichroism, FT-IR and Raman of proteins. Appl Spectrosc 1999, 53:655-665.

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Keiderling TA, Xu Q: Unfolded peptides and proteins studied with infrared absorption and vibrational circular dichroism spectra. In Unfolded Proteins, Adv Prot Chem vol 62. Edited by Rose GD. New York: Academic Press; 2002:111-162. Full discussion of the characteristic VCD of denatured proteins and peptides by thermal and, where possible, chemical means. Correlation of the amide I′ bandshape with that of PLP II demonstrates the propensity to develop a local left-handed twist in an extended conformation. Variation of conditions (temperature) shows a more disordered state to arise, but to be often obscured in proteins by aggregation. 38. Tanaka T, Inoue K, Kodama T, Kyogoku Y, Hayakawa T, Sugeta H: α-phenethyl)-L-glutamate] using Conformational study on poly[γγ-(α vibrational circular dichroism spectroscopy. Biopolymers 2001, 62:228-234. 39. Urbanova M, Setnicka V, Kral V, Volka K: Nonconvalent interaction of peptides with porphyrins in aqueous solution: conformation study using vibrational CD spectroscopy. Biopolymers 2001, 60:307-316. 40. Yasui SC, Keiderling TA: Vibrational circular dichroism of polypeptides. VI. Polytyrosine alpha-helical and random coil results. Biopolymers 1986, 25:5-15. 41. Yasui SC, Keiderling TA, Sisido M: Vibrational circular dichroism of polypeptides XI. Conformation of poly(L-lysine(Z)-L-lysine(Z)L-1-pyrenylalanine) and poly(L-lysine(Z)- L-lysine(Z)-L-1napthylalanine) in solution. Macromolecules 1987, 20:2403-2406. 42. Yasui SC, Keiderling TA, Bonora GM, Toniolo C: Vibrational circular dichroism of polypeptides V. A study of 310 helical octapeptides. Biopolymers 1986, 25:79-89. 43. Yoder G, Polese A, Silva RAGD, Formaggio F, Crisma M, Broxterman QB, Kamphuis J, Toniolo C, Keiderling TA: α-Me)Val Conformational characterization of terminally blocked L-α homopeptides using vibrational and electronic circular dichroism — 310-helical stabilization by peptide–peptide interaction. J Am Chem Soc 1997, 119:10278-10285.

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44. Kubelka J, Silva RAGD, Keiderling TA: Spectroscopic discrimination • between peptide 310- and α-helices. Study of the impact of α-methyl substitution. J Am Chem Soc 2002, 124:5325-5332. VCD and IR of (Ala)2n, (Aib–Ala)n, and (Aib)2n oligomers are calculated for both α- and 310-helical conformations and compared with data for Ala-rich, Aib-rich and Aib–Ala mixed oligomers. The theoretical results qualitatively predict the small intensity shifts with increase in alanine content that occur in the 310 helix form and confirm that the major intensity differences in VCD between α- and 310-helices is not due to αMe substitution.

31. Wang F, Polavarapu PL: Temperature influence on the secondary structure of avidin and avidin-biotin complex: a vibrational circular dichroism study. J Phys Chem 2001, 105:7857-7864.

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Analytical techniques

46. Keiderling TA, Silva RAGD, Yoder G, Dukor RK: Vibrational circular dichroism spectroscopy of selected oligopeptide conformations. Bioorg Med Chem 1999, 7:133-141. Kubelka J, Keiderling TA: Differentiation of β-sheet-forming structures: ab initio-based simulations of IR absorption and vibrational CD for model peptide and protein β-sheets. J Am Chem Soc 2001, 123:12048-12058. Computations of IR and VCD spectra for β-sheets with 2–5 strands with and without twisting shows the ‘characteristic’ amide I β-sheet IR is a property of flat extended sheets, which have very weak VCD, in agreement with experiment. Twisting the sheets leads to significant VCD, as seen in proteins, but parallel or anti-parallel twisted sheets would be difficult to distinguish by IR or VCD. 47. ••

48. Espinosa JF, Munoz V, Gellman SH: Interplay between hydrophobic cluster and loop propensity in β-hairpin formation. J Mol Biol 2001, 306:397-402. 49. Stanger HE, Syud FA, Espinosa JF, Giriat I, Muir T, Gellman SH: Length-dependent stability and strand length limits in antiparallel β-sheet secondary structure. Proc Natl Acad Sci USA 2001, 98:12015-12020. 50. Gellman SH: Minimal model systems for β-sheet secondary structure in proteins. Curr Opin Chem Biol 1998, 2:717-725. 51. Zhao C, Polavarapu PL, Das C, Balaram P: Vibrational circular •• dichroism of β-hairpin peptides. J Am Chem Soc 2000, 122:8228-8231. VCD for a series of β-hairpins in non-polar solution stabilized by D-Pro-Gly turns are presented and compared with NMR ROSEY data showing long range NOEs between the strands and, for phenylalanine-containing variants, proposing an interaction geometry between aromatic side chains to explain their unusual ECD. 52. Hilario J, Kubelka J, Syud FA, Gellman SH, Keiderling TA: Spectroscopic characterization of selected β-sheet hairpin models. Biopolymers 2002, 67:233-263. 53. Xie P, Zhou QW, Diem M: Conformational studies of β-turns in cyclic peptides by vibrational CD. J Am Chem Soc 1995, 117:9502-9508. 54. Xie P, Diem M: Conformational studies of cyclo-(-Pro-Gly-)(3) and its complexes with cations by vibrational circular dichroism. J Am Chem Soc 1995, 117:429-437. 55. Ito H: Linear response polarizability bandshape calculations of vibrational circular dichroism, vibrational absorption, and electronic circular dichroism of cyclo(Gly-Pro-Gly-D-Ala-Pro): a small cyclic pentapeptide having beta- and gamma- turns. Biospectroscopy 1996, 2:17-37. 56. Kubelka J: Vibrational Spectroscopic Studies of Peptide and Protein Structures. Theory and Experiment. Ph.D Thesis, University of Illinois at Chicago, 2002. 57.

Yoder G, Pancoska P, Keiderling TA: Characterization of alanine-rich peptides, Ac-(AAKAA)n-GY-NH2 (n=1–4) using vibrational circular dichroism and Fourier transform infrared. Conformational determination and thermal unfolding. Biochemistry 1997, 36:15123-15133.

58. Decatur SM, Antonic J: Isotope-edited FTIR spectroscopy of helical peptides. J Am Chem Soc 1999, 121:11914-11915. 59. Torres J, Kukol A, Goodman JM, Arkin IT: Site-specific examination of secondary structure and orientation determination in

membrane proteins: the peptidic 13C=18O group as a novel infrared probe. Biopolymers 2001, 59:396-401. 60. Silva RAGD, Kubelka J, Decatur SM, Bour P, Keiderling TA: Site •• specific conformational determination in thermal unfolding studies of helical peptides using vibrational circular dichroism with isotopic substitution. Proc Natl Acad Sci USA 2000, 97:8318-8323. The first application of isotopic substitution to develop site-specific conformational information with VCD. Experimental and theoretical VCD of a series of 20mer peptides with four 13C labels in each were used to determine that while overall α-helical, the C terminus was unwound. VCD analysis showed the terminal residues to be less stable than the central ones, confirming the mechanism of unwinding from the ends with an intermediate formed between helix and coil states proposed earlier [57]. 61. Rohl CA, Baldwin RL: Comparison of NH exchange and circular dichroism as techniques for measuring the parameters of the helix-coil transition in peptides. Biochemistry 1997, 36:8435-8442. 62. Bolin KA, Millhauser GL: Alpha and 3(10): the split personality of polypeptide helices. Acc Chem Res 1999, 32:1027-1033. 63. Bour P, Keiderling TA: Ab initio simulation of the vibrational circular dichroism of coupled peptides. J Am Chem Soc 1993, 115:9602-9607. 64. Bour P, Kubelka J, Keiderling TA: Simulations of oligopeptide vibrational circular dichroism. Effects of isotopic labeling. Biopolymers 2000, 53:380-395. 65. Jalkanen KJ, Suhai S: N-acetyl-L-alanine-N′′-methylamide: a density functional analysis of the vibrational absorption and vibrational circular dichroism spectra. Chem Phys 1996, 208:81-116. 66. Knapp-Mohammady M, Jalkanen KJ, Nardi F, Wade RC, Suhai S: • L-Alanyl-L-alanine in the zwitterionic state: structures determined in the presence of explicit water molecules and with continuum models using density functional theory. Chem Phys 1999, 240:63-77. For a small fully optimized peptide, explicit water effects on the IR and VCD spectra are calculated ab initio and compared with simulations using continuum models for solvent. 67.

Bour P, Sopkova J, Bednarova L, Malon P, Keiderling TA: Transfer of molecular property tensors in Cartesian coordinates: a new algorithm for simulation of vibrational spectra. J Comput Chem 1997, 18:646-659.

68. Kubelka J, Keiderling TA: The anomalous infrared amide I intensity distribution in 13C isotopically labeled peptide β-sheets comes from extended, multiple-stranded structures. An ab initio study. J Am Chem Soc 2001, 123:6142-6150. 69. Bohr HG, Frimand K, Jalkanen KJ, Nieminen RM, Suhai S: Neuralnetwork analysis of the vibrational spectra of N-acetyl L-alanyl N′′-methyl amide conformational states. Phys Rev E 2001, 64:1905. 70. Han WG, Elstner M, Jalkanen KJ, Frauenheim T, Suhai S: Hybrid SCC-DFTB/molecular mechanical studies of H-bonded systems and of N-acetyl-(L-Ala) (n) N′′-methylamide helices in water solution source. Int J Quantum Chem 2000, 78:459-479. 71. Kubelka J, Keiderling TA: Ab initio calculation of amide carbonyl stretch vibrational frequencies in solution with modified basis sets. 1. N-methyl acetamide. J Phys Chem 2001, 105:10922-10928.