Vibrational circular dichroism and IR spectral analysis as a test of theoretical conformational modeling for a cyclic hexapeptide

A model cyclohexapeptide, cyclo-(Phe-(D)Pro-Gly-Arg-Gly-Asp) was synthesized and its IR and VCD spectra were used as a test of density functional theory (DFT) level predictions of spectral intensities for a peptide with a nonrepeating but partially constricted conformation. Peptide structure and flexibility was estimated by molecular dynamics (MD) simulations and the spectra were simulated using full quantum mechanical (QM) approaches for the complete peptide and for simplified models with truncated side chains. After simulated annealing, the backbone conformation of the ring structure is relatively stable, consisting of a normal beta-turn and a tight loop (no H-bond) which does not vary over short trajectories. Only in quite long MD runs at high temperatures do other conformations appear. MD simulations were carried out for the cyclic peptide in water and in TFE, which match experimental solvents, as well as with and without protonation of the Asp carboxyl group. DFT spectral simulations were made using the annealed structure and were extended to include basis set variation, to determine an optimal computational approach, and solvent simulation with a polarized continuum model (PCM). Stepwise full DFT simulation of spectra was done for various sequences with the same backbone geometry but based on (1) solely Gly residues, (2) Ala substitution except Gly and Pro, and (3) complete sequences with side chains. Additionally, a selection of structures was used to compute IR and VCD spectra with the optimal method to determine structural variation effects. The side chains, especially the Asp-COOH and Arg-NH(2) transitions, had an impact on the computed amide frequencies, IR intensities and VCD pattern. Since experimentally these groups would have little chirality, due to conformational variation, they do not impact the observed VCD spectra. Correcting for frequency shifts, the Ala model for the cyclopeptide gives the clearest representation of the amide VCD. The experimental sign pattern for the amide I' band in D(2)O and also the sharper, more intense amide I VCD band in TFE was seen to some degree in one conformer with Type II' turns, but the data favor a mix of structures.     

VCD Robustness of the Amide‐I and Amide‐II Vibrational Modes of Small Peptide Models

The rotational strengths and the robustness values of amide‐I and amide‐II vibrational modes of For(AA)_nNHMe (where AA is Val, Asn, Asp, or Cys, n = 1–5 for Val and Asn; n = 1 for Asp and Cys) model peptides with α‐helix and β‐sheet backbone conformations were computed by density functional methods. The robustness results verify empirical rules drawn from experiments and from computed rotational strengths linking amide‐I and amide‐II patterns in the vibrational circular dichroism (VCD) spectra of peptides with their backbone structures. For peptides with at least three residues (n ≥ 3) these characteristic patterns from coupled amide vibrational modes have robust signatures. For shorter peptide models many vibrational modes are nonrobust, and the robust modes can be dependent on the residues or on their side chain conformations in addition to backbone conformations. These robust VCD bands, however, provide information for the detailed structural analysis of these smaller systems. Chirality 27:625–634, 2015 © 2015 Wiley Periodicals, Inc.     

Solution conformations of cyclosporins and magnesium-cyclosporin complexes determined by vibrational circular dichroism

Vibrational circular dichroism (VCD) spectroscopy was used to investigate the solution conformations of cyclosporins A, C, D, G, and H in CDCl(3), in the amide I and NH/OH-stretching regions, and their corresponding magnesium complexes in CD(3)CN, in the amide I region. VCD spectra are sensitive to the chiral arrangement of Cdbond;O and NH bonds in this cyclic undecapeptide. Calculations of molecular geometries, as well as IR and VCD intensities of model cyclosporin fragments that include the intramolecular hydrogen bonds of the crystal conformations of cyclosporins A and H (CsA and CsH), were carried out at the density functional theory (DFT; BPW91 functional/6-31G* basis set) level. The good agreement between IR and VCD spectra from experiment and DFT calculations provides evidence that the crystal conformation of CsA is dominant in CDCl(3) solution; CsH, however, assumes both an intramolecularly hydrogen-bonded crystal conformation and more open forms in solution. Comparisons of the experimental and calculated VCD spectra in the NH/OH-stretching region of the noncomplexed cyclosporins indicate that conformers with both free and hydrogen-bonded NH and OH groups are present in solution. Differences between the IR and VCD spectra for the metal-free and magnesium-complexed cyclosporins are indicative of strong interactions between cyclosporins and magnesium ions.     

Electronic and vibrational circular dichroism of aromatic amino acids by density functional theory

Structures of model compounds mimicking aromatic amino acid residues in proteins are optimized by density functional theory (DFT), assuming that the main-chain conformation was a random coil. Excitation energies and dipole and rotational strengths for the optimized structures were calculated based on time-dependent DFT (TD-DFT). The electronic circular dichroism (ECD) bands of the models were significantly affected by side-chain conformations. Hydration models of the aromatic residues were also subjected to TD-DFT calculations, and the ECD bands of these models were found to be highly perturbed by the hydration of the main-chain amide groups. In addition to calculating the random-coil conformation, we also performed TD-DFT calculations of the aromatic residue models, assuming that the main-chain conformation was an alpha-helix or beta-strand. As expected, the overall feature of the ECD bands was also perturbed by the main-chain conformations. Moreover, vibrational circular dichroism (VCD) spectra of the hydration models in a random-coil structure were simulated by DFT, which showed that the VCD spectra are more sensitive to the side-chain conformations than the ECD spectra. The present results show that analyses combining ECD and VCD spectroscopy and using DFT calculations can elucidate the main- and side-chain conformations of aromatic residues in proteins.     

Conformational behaviour of Cα,α-diphenylglycine: folded vs. extended structures in DϕG-containing tripeptides

The crystal structures of three fully protected tripeptides containing the Dϕg residue (C^α,α-diphenylglycine) in the central position are reported, namely Z-Gly-Dϕg-Gly-OMe ( a ), Z-Gly-Dϕg-Aib-OMe ( b ) and Z-Aib-Dϕg-Aib-OMe ( c ). The molecular conformations are quite unusual because the Dϕg residue adopts a folded conformation in the 3₁₀-helical region when the following residue adopts a folded conformation of opposite handedness (peptides b and c ). In contrast, the Dϕg residue adopts the more frequently observed fully extended conformation when the following residue adopts a semi-extended conformation (peptide a ). These findings are in agreement with the theoretical calculations on Ac-Dϕg-Aib-NHCH₃ and Ac-Aib-Dϕg-NHCH₃ also reported in this work. © 1998 European Peptide Society and John Wiley & Sons, Ltd.     

Are density functional theory predictions of the Raman spectra accurate enough to distinguish conformational transitions during amyloid formation?

We report density functional theory (DFT) calculations of the Raman spectra for hexapepetides of glutamic acid and lysine in three different conformations (α, β and PPII). The wave numbers of amide I, amide II and amide III bands of all three conformations predicted at B3LYP/6-31G and B3LYP/6-31G* are in good agreement with previously reported experimental values of polyglutamic acid and polylysine. Agreement with experiment improves when polarization functions are included in the basis set. Explicit water molecules, H-bonded to the backbone amide groups were found to be absolutely necessary to obtain this agreement. Our results indicate that DFT is a promising tool for assignment of the spectral data on kinetics of conformational changes for peptides during amyloid formation.     

Synthetic α-helical peptides incorporating intercalators for DNA recognition

The design and synthesis of a water-soluble 14-residue peptide, in which a quinoline intercalator is attached to the peptide backbone via alkylation of a central cysteine residue, is reported. 600 MHz ¹H NMR spectroscopy and circular dichroism indicate that the peptide forms a nascent helix in aqueous solution, ie. an ensemble of turn-like structures over several adjacent residues in the peptide. A large number of sequential d_NN(i, i+1) connectivities were observed in NOESY spectra, and titration of trifluoroethanol into a solution of the peptide resulted in the characteristic CD spectrum expected for an α-helix. At low DNA concentrations, CD spectroscopy indicates that this helical conformation is stabilized, presumably due to folding of the peptide in the major groove of DNA.     

Fourier transform vibrational circular dichroism as a decisive tool for conformational studies of peptides containing tyrosyl residues

Previous UV-circular dichroism (UV-CD) and NMR studies showed that Ac-AAAAAAAEAAKA-NH(2) has an alpha-helical structure in 50% (v/v) aqueous trifluoroethanol. Replacement of Ala(1) to Ala(6) with Tyr results in spectra that show an apparent loss of helicity in the same solvent. This apparent loss of helicity could be attributed to the coupling of the tyrosyl side chain chromophore with the backbone amide. However, such electronic coupling does not affect the vibrational CD (VCD) spectra. The VCD spectra of the peptides with tyrosyl residues were identical to that of the peptide containing no Tyr, which shows the same alpha-helical structure. Because it is now clear that Tyr replacement does not change the backbone conformation of peptides, UV-CD measurements should be complemented by VCD to determine the secondary structure when electronic effects can disturb the UV-CD spectrum of the inherent structure.     

Vibrational and chiroptical spectroscopic characterization of γ‐turn model cyclic tetrapeptides containing two β‐Ala residues

The optical spectroscopic characterization of γ‐turns in solution is uncertain and their distinction from β‐turns is often difficult. This work reports systematic ECD and vibrational circular dichroism (VCD) spectroscopic studies on γ‐turn model cyclic tetrapeptides cyclo(Ala‐β‐Ala‐Pro‐β‐Ala) ( 1 ), cyclo(Pro‐β‐Ala‐Pro‐β‐Ala) ( 2 ) and cyclo(Ala‐β‐Ala‐Ala‐β‐Ala) ( 3 ). Conformational analysis performed at the 6‐31G(d)/B3LYP level of theory using an adequate PCM solvent model predicted one predominant conformer for 1‐3 , featuring two inverse γ‐turns. The ECD spectra in ACN of 1 and 2 are characterized by a negative n→π* band near 230 nm and a positive π→π* band below 200 nm with a long wavelength shoulder. The ECD spectra in TFE of 1‐3 show similar spectra with blue‐shifted bands. The VCD spectra in ACN‐d₃ of 1 and 2 show a +/?/+/? amide I sign pattern resulting from four uncoupled vibrations in the case of 1 and a sequence of two positive couplets in the case of 2 . A ?/+/+/? amide I VCD pattern was measured for 3 in TFE‐d₂. All three peptides give a positive couplet or couplet‐like feature (+/?) in the amide II region. VCD spectroscopy, in agreement with theoretical calculations revealed that low frequency amide I vibrations (at ～1630 cm^?1 or below) are indicative of a C₇ H‐bonded inverse γ‐turns with Pro in position 2, while γ‐turns encompassing Ala absorb at higher frequency (above 1645 cm^?1). Chirality, 2010. © 2010 Wiley‐Liss, Inc.     

Controlling the helical screw sense of peptides with C‐terminal L‐valine

One chiral L ‐valine (L ‐Val) was inserted into the C‐terminal position of achiral peptide segments constructed from α‐aminoisobutyric acid (Aib) and α,β‐dehydrophenylalanine (Δ^ZPhe) residues. The IR, ¹H NMR and CD spectra indicated that the dominant conformations of the pentapeptide Boc‐Aib‐ΔPhe‐(Aib)₂‐L ‐Val‐NH‐Bn (3) and the hexapeptide Boc‐Aib‐ΔPhe‐(Aib)₃‐L ‐Val‐NH‐Bn (4) in solution were both right‐handed (P) 3₁₀‐helical structures. X‐ray crystallographic analyses of 3 and 4 revealed that only a right‐handed (P) 3₁₀‐helical structure was present in their crystalline states. The conformation of 4 was also studied by molecular‐mechanics calculations. Copyright © 2010 European Peptide Society and John Wiley & Sons, Ltd.     

Raman and infrared spectroscopic study of gramicidin A conformations

Raman scattering and infrared spectroscopic techniques were used to study the vibrational spectrum and conformation of the membrane channel protein gramicidin A in the solid state, in organic solutions and, using Raman scattering only, in a phospholipid environment. The investigation also includes measurements on head- and tail-group-modifled gramicidin A and a potassium thiocyanate-gramicidin A complex. Tentative identification of the molecular vibrations is proposed on the basis of the data on model compounds. The existence of four distinct conformations of the gramicidin A chain is established: conformation I present in the solid state, and CH₃OH and CD₃OD solutions; conformation II present in films cast from CHCl₃ solution; conformation III present in (CH₃)₂SO and (CD₃)₂SO solutions at concentrations below 0.5 m gramicidin A; and conformation IV present in the potassium thiocyanate-gramicidin A complex. The data obtainable on a gramicidin A-phospholipid suspension indicate a gramicidin A conformation in this environment corresponding either to the conformation I or II. The details of the spectra in the amide I region are shown to be consistent with a β-parallel hydrogen-bonded π_LD helix for conformational I, in terms of the polypeptide vibrational calculations of Nevskaya and co-workers. Conformation II is found to be consistent with an antiparallel double-stranded π_LD helix, while conformations III and IV probably have π-helical structures with larger channel diameters. The data on head- and tail-modified gramicidin A molecules indicate that their conformations are only slightly different from that of gramicidin A in conformation I.     

VCD spectroscopic investigation of enantiopure cyclic beta-lactams obtained through Lipolase-catalyzed enantioselective ring-opening reaction

A direct enzymatic method for the preparation of cyclic beta-lactams and beta-amino acids was recently developed, involving the Lipolase-catalyzed enantioselective hydrolysis of racemic beta-lactams in an organic solvent. Vibrational circular dichroism (VCD) spectroscopy combined with quantum chemical calculations at ab initio (DFT) level of theory has now been applied to determine the absolute configuration and conformation of a series of cyclic beta-lactams (1-10). The absolute configuration of 8 was derived from X-ray crystallography. Only indirect evidence was available for 1, 2, 5, 6, and 7. The absolute configuration of the new lactams 3, 4, 9, and 10 was not known previously. The VCD analysis indicated the homochirality of the studied lactams. The conformation of the flexible beta-lactams was also predicted from the VCD data. Even in the cases where multiple conformers are allowed, the predominance of one conformer was found, with the exception of 2, being present as a mixture of four conformers. Beta-lactams tend to form H-bonded dimers. The fine structure of the amide I VCD band suggested that only a small population of H-bonded dimers is formed in deuterated chloroform.     

Synthesis and conformational analysis of aib-containing peptide modelling for N-glycosylation site in N-glycoprotein

A tetrapetide containing an Aib residue, Boc-Asn-Aib-Thr-Aib-OMe, was synthesized as a peptide model for the N-glycosylation site in N-glycoproteins. Backbone conformation of the peptide and possible intramolecular interaction between the Asn and Thr side chains were elucidated by means of n.m.r. spectroscopy. Temperature dependence of NH proton chemical shift and NOE experiments showed that Boc-Asn-Aib-Thr-Aib-OMe has a tendency to form a β-turn structure with a hydrogen bond involving Thr and Aib⁴ NH groups. Incorporation of Aib residues in the peptide model promotes folding of the peptide backbone. With folded backbone conformation, carboxyamide protons of the Asn residue are not involved in hydrogen bond network, while the OH group of the Thr residue is a candidate for a hydrogen bond in DMSO-d₆ solution.     

Vibrational circular dichroism as a probe of solid‐state organisation of derivatives of cyclic β‐amino acids: Cis‐ and trans‐2‐aminocyclobutane‐1‐carboxylic acid

Peptide models built from cis‐ and trans‐2‐aminocyclobutane‐1‐carboxylic acids (ACBCs) are studied in the solid phase by combining Fourier‐transform infrared spectroscopy (FTIR) absorption spectroscopy, vibrational circular dichroism (VCD), and quantum chemical calculations using density functional theory (DFT). The studied systems are N‐tert‐butyloxycarbonyl (Boc) derivatives of 2‐aminocyclobutanecarboxylic acid (ACBC) benzylamides, namely Boc?(cis‐ACBC)?NH?Bn and Boc?(trans‐ACBC)?NH?Bn. These two diastereomers show very different VCD signatures and intensities, which of the trans‐ACBC derivative being one order of magnitude larger in the region of the ν (CO) stretch. The spectral signature of the cis‐ACBC derivative is satisfactorily reproduced by that of the monomer extracted from the solid‐state geometry of related ACBC derivatives, which shows that no long‐range effects are implicated for this system. In terms of hydrogen bonds, the geometry of this monomer is intermediate between the C6 and C8 structures (exhibiting a 6‐ or 8‐membered cyclic NH?O hydrogen bond) previously evidenced in the gas phase. The benzyl group must be in an extended geometry to reproduce satisfactorily the shape of the VCD spectrum in the ν (CO) range, which qualifies VCD as a potential probe of dispersion interaction. In contrast, reproducing the IR and VCD spectrum of the trans‐ACBC derivative requires clusters larger than four units, exhibiting strong intermolecular H‐bonding patterns. A qualitative agreement is obtained for a tetramer, although the intensity enhancement is not reproduced. These results underline the sensitivity of VCD to the long‐range organisation in the crystal.     

Structural Basis and Target-specific Modulation of ADP Sensing by the Synechococcus elongatus PII Signaling Protein

P_II signaling proteins comprise one of the most versatile signaling devices in nature and have a highly conserved structure. In cyanobacteria, PipX and N-acetyl-l-glutamate kinase are receptors of P_II signaling, and these interactions are modulated by ADP, ATP, and 2-oxoglutarate. These effector molecules bind interdependently to three anti-cooperative binding sites on the trimeric P_II protein and thereby affect its structure. Here we used the P_II protein from Synechococcus elongatus PCC 7942 to reveal the structural basis of anti-cooperative ADP binding. Furthermore, we clarified the mutual influence of P_II-receptor interaction and sensing of the ATP/ADP ratio. The crystal structures of two forms of trimeric P_II, one with one ADP bound and the other with all three ADP-binding sites occupied, revealed significant differences in the ADP binding mode: at one site (S1) ADP is tightly bound through side-chain and main-chain interactions, whereas at the other two sites (S2 and S3) the ADP molecules are only bound by main-chain interactions. In the presence of the P_II-receptor PipX, the affinity of ADP to the first binding site S1 strongly increases, whereas the affinity for ATP decreases due to PipX favoring the S1 conformation of P_II-ADP. In consequence, the P_II-PipX interaction is highly sensitive to subtle fluctuations in the ATP/ADP ratio. By contrast, the P_II-N-acetyl-l-glutamate kinase interaction, which is negatively affected by ADP, is insensitive to these fluctuations. Modulation of the metabolite-sensing properties of P_II by its receptors allows P_II to differentially perceive signals in a target-specific manner and to perform multitasking signal transduction.     

Toward engineering efficient peptidomimetics. Screening conformational landscape of two modified dehydroaminoacids

Effective peptidomimetics should posses structural rigidity and appropriate interaction pattern leading to potential spatial and electronic matching to the target receptor site. Rational design of such small bioactive molecules could push chemical synthesis and molecular modeling toward faster progress in medicinal chemistry. Conformational properties of N‐t‐butoxycarbonyl‐glycine‐(E/Z)‐dehydrophenylalanine N′,N′‐dimethylamides (Boc‐Gly‐(E/Z)‐ΔPhe‐NMe₂) in chloroform were studied by NMR and IR spectroscopy. The experimental findings were supported by extensive calculations at DFT(B3LYP, M06‐2X) and MP2 levels of theory and the β‐turn tendency for both isomers of the studied dipeptide were determined in vacuum and in solution. The theoretical data and experimental IR results were used as an additional information for the NMR‐based determination of the detailed solution conformations of the peptides. The obtained results reveal that N‐methylation of C‐terminal amide group changes dramatically the conformational properties of studied dehydropeptides. Theoretical conformational analysis reveals that the tendency to adopt β‐turn conformations is much weaker for the N‐methylated Z isomer (Boc‐Gly‐(Z)‐ΔPhe‐NMe₂), both in vacuum and in polar environment. On the contrary, N‐methylated E isomer (Boc‐Gly‐(E)‐ΔPhe‐NMe₂) can easily adopt β‐turn conformation, but the backbone torsion angles (φ₁, ψ₁, φ₂, ψ₂) are off the limits for common β‐turn types. © 2013 Wiley Periodicals, Inc. Biopolymers 101: 28–40, 2014.     

Structure and Thermodynamics of Effector Molecule Binding to the Nitrogen Signal Transduction PII Protein GlnZ from Azospirillum brasilense

The trimeric P_II signal transduction proteins regulate the function of a variety of target proteins predominantly involved in nitrogen metabolism. ATP, ADP and 2-oxoglutarate (2-OG) are key effector molecules influencing P_II binding to targets. Studies of P_II proteins have established that the 20-residue T-loop plays a central role in effector sensing and target binding. However, the specific effects of effector binding on T-loop conformation have remained poorly documented. We present eight crystal structures of the Azospirillum brasilense P_II protein GlnZ, six of which are cocrystallized and liganded with ADP or ATP. We find that interaction with the diphosphate moiety of bound ADP constrains the N-terminal part of the T-loop in a characteristic way that is maintained in ADP-promoted complexes with target proteins. In contrast, the interactions with the triphosphate moiety in ATP complexes are much more variable and no single predominant interaction mode is apparent except for the ternary MgATP/2-OG complex. These conclusions can be extended to most investigated P_II proteins of the GlnB/GlnK subfamily. Unlike reported for other P_II proteins, microcalorimetry reveals no cooperativity between the three binding sites of GlnZ trimers for any of the three effectors under carefully controlled experimental conditions.     

Vibrational Circular Dichroism Spectra of Proteins in the Amide III Region: Measurement and Correlation of Bandshape to Secondary Structure

Vibrational circular dichroism (VCD) spectra have been measured for 23 globular proteins dissolved in H₂O/phosphate buffer over the 1400 to 1100 cm⁻¹region which encompasses the amide III mode. Spectral responses characteristic of the dominant secondary structure type were found as broad features at ∼1300 cm⁻¹, with the extreme forms having positive VCD for highly helical proteins and negative VCD for highly sheet-containing proteins. Quantitative correlation with secondary structure was carried out using previously developed factor analysis and restricted multiple regression (FA/RMR) techniques. Since the absorbance intensity of the amide III mode is difficult to determine due to overlap with other transitions, an alternative, absolute intensity-independent, simple structural analysis method was used. A linear regression was developed between the fractional components of secondary structure for the protein set and the overlap integrals of the normalized spectra from the set with that of a selected protein. The results of this simple method are quite comparable to those of the FA/RMR approach for analysis with amide III VCD. On the other hand, test calculations with the new method when used with electronic CD spectra are not as good as FA/RMR due to its more intensity-dependent relationship with secondary structure.     

Structures of intact glycoproteins from vibrational circular dichroism

Vibrational circular dichroism (VCD) spectra for the glycoproteins alpha1-acid glycoprotein (AGP) and bovine submaxillary mucin (BSM), have been measured in D2O solutions and for the films prepared from aqueous (H2O) buffer solutions in the 1800 to 900 cm(-1) region. The solution VCD results revealed that AGP has beta-sheet structure, along with a significant amount of alpha-helix as evidenced from a W pattern in the amide I region. The VCD of BSM solution suggested a polyproline II type structure, characterized by the appearance of strong negative couplet in the amide I region. The film VCD results on AGP and BSM suggested that the secondary structures of polypeptide fold in the film state are similar to those in the solution. The absence of any significant film VCD in the low frequency region (1200-900 cm(-1)), suggested that the dominant linkage for carbohydrate residues is likely to be a beta linkage. VCD spectroscopy gains importance in the secondary structural analysis of polypeptide fold in glycoproteins due to the absence of interfering VCD from the carbohydrate residues in the conformationally sensitive amide I region. Also, film VCD studies permit measurements in the low wavenumber region (1200-900 cm(-1)) that reveal the dominant type of linkage for carbohydrate residues. Such clear structural information is unlike that from ECD, where ECD bands of acylated amino sugar residues interfere with those of polypeptide backbone in the conformationally sensitive far-UV region.     

Low concentration structural dynamics of lanreotide and somatostatin‐14

Lanreotide, a synthetic cyclic octapeptide, analogue of the peptide hormone somatostatin‐14 (SST‐14), is routinely used as a long‐acting medication in the management of neuroendocrine tumors. Despite its therapeutic importance, low concentration structural data is still lacking for lanreotide. In fact, the major part of the previous structural investigations were focused on the remarkable aggregation properties of this peptide, appearing at high concentrations (>5 mM). Here, we have applied three optical spectroscopic techniques, i.e. fluorescence, circular dichroism and Raman scattering, for analyzing the structural dynamics at the concentrations below 5 mM, where lanreotide exists either in a monomer state or at the first stages of aggregation. The obtained data from lanreotide were discussed through their comparison with those collected from SST‐14, leading us to the following conclusions: (i) The central D‐Trp residue, forming with its adjacent Lys the main receptor interacting part of lanreotide, keeps a constant high rotational freedom whatever the environment (water, water/methanol, methanol). (ii) A solvent‐dependent tight β‐turn, belonging to the type‐II' family, is revealed in lanreotide. (iii) Raman data analyzed by band decomposition in the amide (I and III) regions allowed estimation of different secondary structural elements within the millimolar range. Interestingly, the applied protocol shows a perfect agreement between the structural features provided by the amide I and amide III Raman markers. © 2014 Wiley Periodicals, Inc. Biopolymers 101: 1019–1028, 2014.