New Fourier transform infrared based computational method for peptide secondary structure determination. II. Application to study of peptide fragments reproducing processing site of ocytocin-neurophysin precursor

A new method for the quantitative determination of the percentage of intramolecular H-bonds, based on Fourier transform infrared techniques, is applied to the conformational analysis of a series of synthetic peptides spanning the processing site of the ocytocin and neurophysin precursor. Even though the method uses traditional tools such as Fourier self-deconvolution, the Nth derivative, and curve-fitting procedures for the analysis of the spectra, the assignment of the absorptions due to peptide groups participating into secondary structures is based on the direct observation and quantification of the isotopic effect induced on the groups participating in intramolecular H-bonds in the presence of organic solvents. This permits the quantification of the different populations of molecules containing intramolecular H-bonds involved in beta-turns and alpha-helices. The results are consistent with those previously obtained by NMR techniques in the same solvent systems.     

Determination of the secondary structure of isomeric forms of human serum albumin by a particular frequency deconvolution procedure applied to fourier transform IR analysis

A new deconvolution procedure was applied to the analysis of Fourier transform in spectra of human serum albumin secondary structure in the native state and in states denatured by heat and acid treatment. The deconvolution method is based on the use of the Conjugate Gradient Minimization Algorithm, with the addition of suitable constraints directly obtained by the application to the measured spectrum of the second derivative operator. This method computes central band frequency, bandwidth, and amplitude of the different spectral components of conformation-sensitive amide bands. In the specific case, it was applied to analysis of the amide I band, and the quantitative determination of the different secondary structures (α-helix, β-sheet, β-turns, and random) was attempted for all the samples examined. The precision of the quantitative determination depends on the amounts of these structures present in the protein. The coefficient of variation is <10% for values of amide I component >15%. The accuracy was tested by comparing, by means of linear regression, the results obtained for human serum albumin, hemoglobin, α-chymotrypsin, and cytochrome c, using our method, with those obtained by x-ray crystallography and CD; the results obtained by other vibrational spectroscopic approaches were also compared. The fit standard error between x-ray and ir secondary structure values estimated by our method is 2.5% for α-helix, 7.16% for β structures, and 5.1% for other structures (turns and random coils). Quantitative results are given for the secondary structures (α-helix, turns, and β-strands) present in the native state (turns and β-strands up to now unknown in aqueous solution), together with the percentages of these structures and additional ones (random coils and β-sheets) formed during denaturization. © 1996 John Wiley & Sons, Inc.     

Protein secondary structures in water from second-derivative amide I infrared spectra

Infrared spectra have been obtained for 12 globular proteins in aqueous solution at 20 degrees C. The proteins studied, which vary widely in the relative amounts of different secondary structures present, include myoglobin, hemoglobin, immunoglobulin G, concanavalin A, lysozyme, cytochrome c, alpha-chymotrypsin, trypsin, ribonuclease A, alcohol dehydrogenase, beta 2-microglobulin, and human class I major histocompatibility complex antigen A2. Criteria for evaluating how successfully the spectra due to liquid and gaseous water are subtracted from the observed spectrum in the amide I region were developed. Comparisons of second-derivative amide I spectra with available crystal structure data provide both qualitative and quantitative support for assignments of infrared bands to secondary structures. Band frequency assignments assigned to alpha-helix, beta-sheet, unordered, and turn structures are highly consistent among all proteins and agree closely with predictions from theory. alpha-Helix and unordered structures can each be assigned to only one band whereas multiple bands are associated with beta-sheets and turns. These findings demonstrate a method of analysis of second-derivative amide I spectra whereby the frequencies of bands due to different secondary structures can be obtained. Furthermore, the band intensities obtained provide a useful method for estimating the relative amounts of different structures.     

Effects of ELF magnetic field on membrane protein structure of living HeLa cells studied by Fourier transform infrared spectroscopy

The effects of exposure to a 50 Hz magnetic field (maximum of 41.7 to 43.6 mT) on the membrane protein structures of living HeLa cells were studied using attenuated total reflection infrared spectroscopy. One min of such exposure shifted peak absorbance of the amide I band to a smaller wave number, reduced peak absorbance of the amide II band, and increased absorbance at around 1600 cm(-1). These results suggest that exposure to the ELF magnetic field has reversible effects on the N-H inplane bending and C-N stretching vibrations of peptide linkages, and changes the secondary structures of alpha-helix and beta-sheet in cell membrane proteins.     

Molecular dynamics simulations of a guaiacyl beta-O-4 lignin model compound: examination of intramolecular hydrogen bonding and conformational flexibility

The dynamical conformational behavior of a guaiacyl beta-O-4 lignin model compound has been investigated by molecular simulations. The potential energy surface of the molecule in vacuum has been examined by means of an adiabatic map, showing a large accessible conformational space with multiple energy minima separated by low barriers. Molecular dynamics simulations have been performed in vacuum and with explicit solvent molecules for 10 and 2.1 ns, respectively. Molecular dynamics trajectories recorded in vacuum have shown the molecule to be flexible and to visit a large number of conformations. Many intramolecular H-bonds have been observed, existing for more than 90% of the total simulation time. The presence of explicit solvent molecules induces a significant broadening of some regions of the accessible conformational space and also largely reduces the statistical significance of intramolecular H-bonding. Intramolecular H-bonds observed in vacuum do not persist significantly and are preferentially exchanged with intermolecular H-bonds to the surrounding solvent molecules. The theoretical results are in good agreement with experimental NMR data that do not support the existence of strong and persistent intramolecular H-bonds in solution but instead indicate that H-bonds to solvent predominate. Finally, both molecular modeling and NMR approaches predict the guaiacyl beta-O-4 structure to be flexible and indicate that intramolecular H-bonds are not strong and persistent enough to confer rigidity to the molecule in solution.     

Quantitative analysis of nucleic acids, proteins, and viruses by Raman band deconvolution

A constrained, iterative Fourier deconvolution method is employed to enhance the resolution of Raman spectra of biological molecules for quantitative assessment of macromolecular secondary structures and hydrogen isotope exchange kinetics. In an application to the Pf1 filamentous bacterial virus, it is shown that the Raman amide I band contains no component other than that due to alpha-helix, indicating the virtual 100% helicity of coat proteins in the native virion. Comparative analysis of the amide I band of six filamentous phages (fd, If1, IKe, Pf1, Xf, and Pf3), all at the same experimental conditions, indicates that the subunit helix-percentage ranges from a high of 100% in Pf1 to a low of 71% in Xf. Deconvolution of amide I of Pf3 at elevated temperatures, for which an alpha-to-beta transition was previously reported (Thomas, G. J., Jr., and L. A. Day, 1981, Proc. Natl. Acad. Sci. USA., 78:2962-2966), allows quantitative evaluation of the contributions of both alpha-helix and beta-strand conformations to the structure of the thermally perturbed viral coat protein. Weak Raman lines of viral DNA bases and coat protein side chains, which are poorly resolved instrumentally, are also distinguished for all viruses by the deconvolution procedure. Application to the carbon-8 hydrogen isotope exchange reaction of a purine constituent of transfer RNA permits accurate determination of the exchange rate constant, which is in agreement with calculations based upon curve-fitting methods.     

Conformational investigation of alpha,beta-dehydropeptides. XVI. Beta-turn tendency in Ac-Pro-DeltaXaa-NHMe: crystallographic and theoretical studies.

The crystal structures of two diastereomeric alpha,beta-dehydrobutyrine peptides Ac-Pro-(Z)-DeltaAbu-NHMe (I) and Ac-Pro-(E)-DeltaAbu-NHMe (II) have been determined. Both dehydropeptides adopt betaI-turn conformation characterized by the pairs of (phi(i+1), psi(i+1)) and (phi(i+2), psi(i+2)) angles as -66, -19, -97, 11 degrees for I and -59, -27, -119, 29 degrees for II. In each peptide, the betaI turn is stabilized by (i + 3) --> i intramolecular hydrogen bonds with N...O distance of 3.12 A for I and 2.93 A for II. These structures have been compared to the crystal structures of homologous peptides Ac-Pro-DeltaVal-NHMe and Ac-Pro-DeltaAla-NHMe. Theoretical analyses by DFT/B3LYP/6-31 + G** method of conformers formed by these four peptides and by the saturated peptide Ac-Pro-Ala-NHMe revealed that peptides with a (Z) substituent at the C(beta) (i+2) atom of dehydroamino acid, i.e. Ac-Pro-DeltaVal-NHMe and Ac-Pro-(Z)-DeltaAbu-NHMe, predominantly form beta turns, both in vacuo and in polar environment. The tendency to adopt beta-turn conformation is much weaker for the peptides lacking the (Z) substituent, Ac-Pro-(E)-DeltaAbu-NHMe and Ac-Pro-DeltaAla-NHMe. The latter adopts a semi-extended or an extended conformation in every polar environment, including a weakly polar solvent. The saturated peptide Ac-Pro-Ala-NHMe in vacuo prefers a beta-turn conformation, but in polar environment the differences between various conformers are small. The role of pi-electron correlation and intramolecular hydrogen bonds interaction in stabilizing the hairpin structures are discussed.     

Effects of organic solutes on the Raman spectra of barnacle muscle fibers

The Raman spectra observed from barnacle muscle fibers are quite complex because the cytoplasm of these cells contains several proteins and solutes. An extraction procedure was used to separate organic solutes from the contractile proteins. Glycine, trimethylamine oxide, taurine, and alanine were found to contribute to the Raman spectra of barnacle muscle fibers, while spectra of lobster fibers reveal the presence of betaine in addition. We have observed that the increase in osmolarity of the intracellular fluid caused by the augmentation of the salinity of sea water (density, 1.023-1.030) in which the barnacles were kept, induces a reduction of intensity of the amide I band. To distinguish among the different parameters which are modified by the sea water salinity, observations were made on glycerinated barnacle muscle fibers. The reduction of intensity of the amide I band in the Raman spectra of glycerinated muscle fibers was also observed with the addition of taurine (0.08 M) in the external relaxing solution. Therefore, under these experimental conditions, the Raman scattering intensity in the amide I region assigned to the alpha-helix conformation (1645-1650 cm-1) is increased when the concentration of organic electrolytes is reduced. However, as no significant decrease of the scattering intensity in the 1660-1670 cm-1 region where the amide I bands of either beta-sheet or disordered conformations normally appear was observed, the increase of intensity of the amide I band centered at 1645 cm-1 is assigned to a change of orientation of alpha-helical segments of the myosin molecules. Our results suggest that organic solutes influence the position of the S-2 segments relative to the thick filaments.     

Redox-induced conformational changes in plastocyanin: an infrared study.

The conformational changes associated with the redox transition of plastocyanin (PC) were investigated by absorption and reaction-induced infrared spectroscopy. In addition to spectral features readily ascribed to beta and turn protein secondary structures, the amide I band shows a major component band at 1647 cm(-1) in both redox states of the protein. The sensitivity of this component to deuteration and increasing temperature suggests that PC adopts an unusual secondary structure in solution, which differs from those described for other type I copper proteins, such as azurin and halocyanin. The conformations of oxidized and reduced PC are different, as evidenced (1) by analysis of their amide I band contour and the electrochemically induced oxidized-minus-reduced difference spectrum and (2) by their different thermal stability. The redox-induced difference spectrum exhibits a number of difference bands within the conformationally sensitive amide I band that could be assigned to peptide C=O modes, in light of their small shift upon deuteration, and to signals attributable to side chain vibrational modes of Tyr residues. Lowering the pH to 4.8 induces destabilization of both redox states of the protein, more pronounced for reduced PC, without significantly affecting their secondary structure. Besides the conformational differences obtained at neutral pH, the oxidized-minus-reduced difference spectrum shows two broad and strong negative bands at 1405 and 1571 cm(-1), assigned to COO(-) vibrations, and a broad positive band at 1710 cm(-1), attributed to the C=O vibration of a COOH group(s). These bands are indicative of a protonation of (an) Asp or Glu side chain(s) upon plastocyanin oxidation at acidic pH.     

Determination of secondary structure of normal fibrin from human peripheral blood

The secondary structure of human fibrin from normal donors and from bovine and suilline plasma was studied by Fourier transform ir spectroscopy and a quantitative analysis of its secondary structure was suggested. For this purpose, a previously experimented spectrum deconvolution procedure based on the use of the Conjugate Gradient Minimisation Algorithm with the addition of suitable constraints was applied to the analysis of conformation-sensitive amide bands. This procedure was applied to amide I and III analysis of bovine and suilline fibrin, obtained industrially, and to amide III analysis of human fibrin clots. The analysis of both amide I and III in the first case was useful in order to test the reliability of the method. We found bovine, suilline, and human fibrin to contain about 30% α-helix (amide I and III components at 1653 cm⁻¹, and 1312 and 1284 cm⁻¹, respectively), 40% β-sheets (amide I and III components at 1625 and 1231 cm⁻¹, respectively) and 30% turns (amide I and III components at 1696, 1680, 1675 cm⁻¹, and 1249 cm⁻¹, respectively). The precision of the quantitative determination depends on the amount of these structures in the protein. Particularly, the coefficient of variation is < 10% for percentage values of amide I and III components > 15 and 5%, respectively. The good agreement of our quantitative data, obtained separately by amide I and amide III analysis, and consistent with a previous fibrinogen (from commercial sources) study that reports only information about fibrin β-sheet content obtained by factor analysis, leads us to believe that the amounts of secondary structures found (α-helix, β-sheets, and turns) are accurate. © 1997 John Wiley & Sons, Inc. Biopoly 41: 545–553, 1997.     

Comparative study of SP[6-11] and its analogs using simulated annealing

In this study we compared the steric structures of the bioactive part of substance P (SP[6-11]) and its analogs (NY3460 and pHOPA-SP5). The molecular dynamics-simulated annealing method was used to explore the conformational space, and the structural differences and similarities of these molecules were identified. For the three peptides, the conformational distributions were represented in Ramachandran density plots. The occurring secondary structural elements of the investigated molecules were identified, namely alpha-Helix, type III beta-Turn, gamma-Turn, and inverse gamma-Turn. For SP[6-11] and its two analogs, different intramolecular interactions (H-bonds between the main-chain atoms, aromatic-aromatic interactions, and amino-aromatic interactions) that can stabilize the various conformations of the three peptides were investigated. Detailed examination of these intramolecular interactions revealed that H-bonds between the main-chain atoms are relevant in the determination and stabilization of the conformer structures of the peptides, while the aromatic-aromatic interactions do not play an important stabilizing role. Furthermore, in the conformers of NY3460 and pHOPA-SP5, different types of amino-aromatic interactions were identified that contribute to the formation of the various structures of these peptides. For all three molecules, the orientations of the side chains were investigated and the rotamer populations were determined.     

Fourier transform infrared spectrometric analysis of protein conformation: effect of sampling method and stress factors.

Changes in the amide bands in Fourier transform infrared spectra of proteins are generally attributed to alterations in protein secondary structure. In this study spectra of five different globular proteins were compared in the solid and solution states recorded with several sampling techniques. Spectral differences for each protein were observed between the various sampling techniques and physical states, which could not all be explained by a change in protein secondary structure. For example, lyophilization in the absence of lyoprotectants caused spectral changes that could (partially) have been caused by the removal of hydrating water molecules rather than secondary structural changes. Moreover, attenuated total reflectance spectra of proteins in H2O were not directly comparable to transmission spectra due to the anomalous dispersion effect. Our study also revealed that the amide I, II, and III bands differ in their sensitivities to changes in protein conformation: For example, strong bands in the region 1620-1630 and 1685-1695 cm(-1) were seen in the amide I region of aggregated protein spectra. Surprisingly, absorbance of such magnitudes was not observed in the amide II and III region. It appears, therefore, that only the amide I can be used to distinguish between intra- and intermolecular beta-sheet formation. Considering the differing sensitivity of the different amide modes to structural changes, it is advisable to utilize not only the amide I band, but also the amide II and III bands, to determine changes in protein secondary structure. Finally, it is important to realize that changes in these bands may not always correspond to secondary structural changes of the proteins.     

Conformational studies of sphingolipids by NMR spectroscopy. I. Dihydrosphingomyelin

The conformational features of dihydrosphingomyelin (DHSM), the major phospholipid of human lens membranes, were investigated by 1H and 31P nuclear magnetic resonance spectroscopy. Several postulates emerge from the observed trends: (a) in partially hydrated samples of DHSM in CDCl3 above 13 mM, at which lipid-lipid interactions prevail, the amide proton is mostly involved in intermolecular H-bonds that link neighboring phospholipids through bridging water molecules. In the absence of water, the NH group is involved in an intramolecular H-bond that restricts the mobility of the phosphate group. (b) In the monomeric form of the lipid molecule, the amide proton of the major conformer is bound intramolecularly with one of the anionic and/or ester oxygens of the phosphate group. A minor conformer may also be present in which the NH proton participates in an intramolecular H-bond linking to the OH group of the sphingoid base. (c) Complete hydration leads to an extension of the head group as water molecules bind to the phosphate and NH groups via H-bonds, thus disrupting the intramolecular H-bonds prevalent at low concentrations.     

Studies of peptides forming 3(10)- and alpha-helices and beta-bend ribbon structures in organic solution and in model biomembranes by Fourier transform infrared spectroscopy

In order to examine the potential correlation between infrared absorption spectra and 3(10)- and alpha-helices and beta-bend ribbon structures, the secondary structures of synthetic peptides known to contain pure 3(10)-helices, mixed 3(10)/alpha-helices, and pure beta-bend ribbon structures, based upon X-ray diffraction and NMR studies, have been investigated by using FTIR spectroscopy incorporating resolution-enhancement techniques. Studies of the peptides known to contain a stable 3(10)-helix in CDCl3 show the main amide I band of fully stable 3(10)-helices occurs at 1666-1662 cm-1. Resolution-enhancement methods revealed small contributions at 1681-1678 and 1646-1644 cm-1, while the amide II band occurs at 1533-1531 cm-1. Peptides known to contain both alpha- and 3(10)-helices in their structure exhibit bands characteristic of both types of conformation. Peptides known to fold into the beta-bend ribbon structure show an amide I band maximum at 1648-1645 cm-1 with the amide II band at 1538-1536 cm-1. Incorporation of these peptides into model membrane structures, e.g., DMPC vesicles, in aqueous buffer sometimes produces changes in the peptide secondary structure. Those peptides which possess a 3(10)-helical structure in CDCl3 solution change the secondary structure in DMPC vesicles to predominantly alpha-helical, plus a contribution from short, unstable 3(10)-helix and/or beta-turns. Those peptides which contain a combination of alpha- and 3(10)-helical structures in CDCl3 solution tend to retain some 3(10)-helical structure within the lipid environment, although the overall H-bonding pattern is altered. Those peptides which form a beta-bend ribbon structure appear to be largely unaffected in the membrane environment.(ABSTRACT TRUNCATED AT 250 WORDS)     

Study of amide-proton exchange of Escherichia coli melibiose permease by attenuated total reflection-Fourier transform infrared spectroscopy: evidence of structure modulation by substrate binding.

The accessibility of Escherichia coli melibiose permease to aqueous solvent was studied following hydrogen-deuterium exchange kinetics monitored by attenuated total reflection-Fourier transform infrared spectroscopy under four distinct conditions where MelB forms different complexes with its substrates (H(+), Na(+), melibiose). Analysis of the amide II band upon (2)H(2)O exposure discloses a significant sugar protection of the protein against aqueous solvent, resulting in an 8% less exchange of the corresponding H(+)*melibiose*MelB complex compared with the protein in the absence of sugar. Investigation of the amide I exchange reveals clear substrate effects on beta-sheet accessibility, with the complex H(+)*melibiose*MelB being the most protected state against exchange, followed by Na(+)*melibiose*MelB. Although of smaller magnitude, similar changes in alpha-helices plus non-ordered structures are detected. Finally, no differences are observed when analyzing reverse turn structures. The results suggest that sugar binding induces a remarkable compactness of the carrier's structure, affecting mainly beta-sheet domains of the transporter, which, according to secondary structure predictions, may include cytoplasmic loops 4-5 and 10-11. A possible catalytic role of these two loops in the functioning of MelB is hypothesized.     

A holistic approach for protein secondary structure estimation from infrared spectra in H(2)O solutions

We present an improved technique for estimating protein secondary structure content from amide I and amide III band infrared spectra. This technique combines the superposition of reference spectra of pure secondary structure elements with simultaneous aromatic side chain, water vapor, and solvent background subtraction. Previous attempts to generate structural reference spectra from a basis set of reference protein spectra have had limited success because of inaccuracies arising from sequential background subtractions and spectral normalization, arbitrary spectral band truncation, and attempted resolution of spectroscopically degenerate structure classes. We eliminated these inaccuracies by defining a single mathematical function for protein spectra, permitting all subtractions, normalizations, and amide band deconvolution steps to be performed simultaneously using a single optimization algorithm. This approach circumvents many of the problems associated with the sequential nature of previous methods, especially with regard to removing the subjectivity involved in each processing step. A key element of this technique was the calculation of reference spectra for ordered helix, unordered helix, sheet, turns, and unordered structures from a basis set of spectra of well-characterized proteins. Structural reference spectra were generated in the amide I and amide III bands, both of which have been shown to be sensitive to protein secondary structure content. We accurately account for overlaps between amide and nonamide regions and allow different structure types to have different extinction coefficients. The agreement between our structure estimates, for proteins both inside and outside the basis set, and the corresponding determinations from X-ray crystallography is good.     

External reflection FTIR of peptide monolayer films in situ at the air/water interface: experimental design, spectra-structure correlations, and effects of hydrogen-deuterium exchange.

A Fourier transform infrared spectrometer has been interfaced with a surface balance and a new external reflection infrared sampling accessory, which permits the acquisition of spectra from protein monolayers in situ at the air/water interface. The accessory, a sample shuttle that permits the collection of spectra in alternating fashion from sample and background troughs, reduces interference from water vapor rotation-vibration bands in the amide I and amide II regions of protein spectra (1520-1690 cm-1) by nearly an order of magnitude. Residual interference from water vapor absorbance ranges from 50 to 200 microabsorbance units. The performance of the device is demonstrated through spectra of synthetic peptides designed to adopt alpha-helical, antiparallel beta-sheet, mixed beta-sheet/beta-turn, and unordered conformations at the air/water interface. The extent of exchange on the surface can be monitored from the relative intensities of the amide II and amide I modes. Hydrogen-deuterium exchange may lower the amide I frequency by as much as 11-12 cm-1 for helical secondary structures. This shifts the vibrational mode into a region normally associated with unordered structures and leads to uncertainties in the application of algorithms commonly used for determination of secondary structure from amide I contours of proteins in D2O solution.     

A holistic approach to protein secondary structure characterization using amide I band Raman spectroscopy

We have developed a holistic protein structure estimation technique using amide I band Raman spectroscopy. This technique combines the superposition of reference spectra for pure secondary structure elements with simultaneous aromatic, fluorescence, and solvent background subtraction, and is applicable to solution, suspension, and solid protein samples. A key component of this technique was the calculation of the reference spectra for ordered helix, unordered helix, and sheet, turns, and unordered structures from a series of well-characterized reference proteins. We accurately account for the overlap between the amide I and non-amide I regions and allow for different scattering efficiencies for different secondary structures. For hydrated samples, we allowed for the possibility that bound water spectra differ from the bulk water spectra. Our computed reference spectra compare well with previous experimental and theoretical results in the literature. We have demonstrated the use of these reference spectra for the estimation of secondary structures of proteins in solution, suspension, and dry solid forms. The agreement between our structure estimates and the corresponding determinations from X-ray crystallography is good.     

Secondary structure of streptokinase in aqueous solution: a Fourier transform infrared spectroscopic study.

The secondary structure of streptokinase (Sk) in aqueous solution was quantitatively examined by using Fourier transform infrared (FT-IR) spectroscopy. Resolution enhancement techniques, including Fourier deconvolution and derivative spectroscopy, were combined with band curve-fitting procedures to quantitate the spectral information from the amide I bands. Nine component bands were found under the broad, nearly featureless amide I bands which reflect the presence of various substructures. The relative areas of these component bands indicate an amount of beta-sheet between 30 and 37% and an alpha-helix content of only 12-13% in Sk. Further conformational substructures are assigned to turns (25-26%) and to "random" structures (15-16%). Additionally, the correlation of a pronounced component band near 1640 cm-1 (10-16% fractional area) with the possible presence of 3(10)-helices is discussed.     

Infrared spectroscopy of collagen and collagen-like polypeptides.

The set of synthetic polytripeptides and polyhexapeptides which can adopt a triple-helical form constitute a good model system for investigating collagen structure. Here we consider previous and new infrared spectroscopic studies on collagen and present the infrared spectra of a number of polymers with collagen-like features. The amide A band position for all triple-helical polypeptides is higher than that observed for most proteins and polypeptides, and this high frequency appears to be related to the degree of supercoiling of the triple helix. It is possible that with increased supercoiling of the three chains the angles between the groups involved in the intramolecular hydrogen bonds become less favorable, or these bonds may become unusually long. The frequency of the amide I band varies considerably for triple-helical polypeptides with different amino acid sequences, and often minor bands are observed. This finding contrasts with the observations for polypeptides in a pleated sheet or α-helical form, where the same amide I frequency is observed regardless of the amino acid composition. An explanation for this variation is proposed in terms of the hydrogen bonding properties of imino acids. Significant spectral changes in the amide I region are observed on hydration in the spectra of some triple-helical polypeptides, but corresponding changes have not been found in the collagens examined.