Vibrational circular dichroism spectra of three conformationally distinct states and an unordered state of poly(L-lysine) in deuterated aqueous solution

Fourier transform ir vibrational circular dichroism (VCD) spectra in the amide I′ region of poly(L-lysine) in D₂O solutions have confirmed the existence of three distinct conformational states and an unordered conformational state in this homopolypeptide. Characteristic VCD spectra are presented for the right-handed α-helix, the antiparallel β-sheet, an extended helix conformation previously referred to as the so-called “random coil,” and a completely unordered conformation characterized by the absence of any amide I′ VCD. VCD for the antiparallel β-sheet in solution and the unordered chain conformation are presented for the first time. Each of the four different VCD spectra is unique in appearance and lends weight to the view that VCD has the potential to become a sensitive new probe of the secondary structure of proteins in solution.     

Vibrational circular dichroism in amino acids and peptides. 7. Amide stretching vibrations in polypeptides

Vibrational circular dichroism (VCD) spectra for the principal amide stretching vibrations, amide A (N? H stretch) and amide I (predominantly C?O stretch), are presented and analyzed for a variety of polypeptides dissolved in chloroform, as well as for two examples in D₂O. Our results for poly(γ-benzyl-L -glutamate) confirm the first and only previous report of VCD in polypeptides carried out by Singh and Keiderling [(1981) Biopolymers 20 , 237–240]. Collectively, our spectra show that the sense of the bisignate VCD in these two regions depends on the sense of α-helicity and not on the absolute configuration of the constituent amino acids. This conclusion is established by obtaining VCD for the two polypeptides, poly(β-benzyl-L -asparate) and poly(im-benzyl-L -histidine), that form left-handed as opposed to right-handed α-helices. A new amide band having significant VCD intensity owing to its Fermi resonance interaction with the N? H stretching mode has been identified as a weak shoulder on the low-frequency side of the amide A band near 3200 cm^?1 and is assigned as a combination band of the amide I and amide II vibrations. VCD spectra of polypeptides in D₂O solution, although weak, have been successfully measured in the amide I region, where spectra appear to be more complicated due to the presence of solvated and internally hydrogen-bonded amide groups. Strong monosignate contributions to the VCD in the amide A and amide I regions for some of the polypeptides indicate coupling of an electronic nature between these two regions and is deduced by an application of the concept of local sum rules of rotational strength. It appears that a detailed understanding of the VCD obtained for polypeptides will not only be diagnostic of secondary structure, but also of more subtle structural and vibrational effects that give rise to local, intrinsic chirality in the amide vibrations.     

Vibrational circular dichroism of polypeptides. VI. Polytyrosine alpha-helical and random-coil results

We have measured the VCD of polytyrosine in the amide I and II regions in dimethyl sulfoxide (DMSO) and in 80:20 mixtures of DMSO with trifluoroethanol, trifluoroacetic acid (TFA), and dichloroacetic acid and in 50:50 mixtures of DMSO and trimethyl phosphate (TMP). Additionally, VCD was obtained for deuterated polytyrosine in DMSO and DMSO:D₂O, DMSO:TFA(d₁), and DMSO:TMP mixtures as before. Amide A VCD was obtained in DMSO and DMSO:TMP mixtures. In the pure solvent, VCD of an opposite sign was seen as compared with that seen in the mixtures. The latter were characteristic in sign pattern and shape of right-handed α-helices for poly(L -tyrosine). The pure polytyrosine:DMSO results are similar to those of polylysine:D₂O at neutral pH and poly(β-benzyl-aspartate) in DMSO and may be characteristic of random-coil VCD.     

The amide III vibrational circular dichroism band as a probe to detect conformational preferences of alanine dipeptide in water

The conformational preferences of blocked alanine dipeptide (ADP), Ac‐Ala‐NHMe, in aqueous solution were studied using vibrational circular dichroism (VCD) together with density functional theory (DFT) calculations. DFT calculations of three most representative conformations of ADP surrounded by six explicit water molecules immersed in a dielectric continuum have proven high sensitivity of amide III VCD band shape that is characteristic for each conformation of the peptide backbone. The polyproline II (P_II) and α_R conformation of ADP are associated with a positive VCD band while β conformation has a negative VCD band in amide III region. Knowing this spectral characteristic of each conformation allows us to assign the experimental amide III VCD spectrum of ADP. Moreover, the amide III region of the VCD spectrum was used to determine the relative populations of conformations of ADP in water. Based on the interpretation of the amide III region of VCD spectrum we have shown that dominant conformation of ADP in water is P_II which is stabilized by hydrogen bonded water molecules between CO and NH groups on the peptide backbone. © 2014 Wiley Periodicals, Inc. Biopolymers 101: 814–818, 2014.     

Vibrational circular dichroism spectra of unblocked proline oligomers.

Vibrational Circular Dichroism (VCD) spectra of unblocked L-proline oligopeptides, (Pro)n n = 3 to 7, dissolved in D2O are reported. For these oligomers, the VCD spectra can be attributed to a conformational dominance of the trans amide conformation with subunits interrelated by a left-handed twist, particularly for the longer oligomers. As a function of oligomer length, formation of this conformation starts at n = 3; and by n = 5 a spectrum closely resembling that of the poly-L-proline II helix in shape and magnitude is seen. The VCD data are compared with previous (Pro)n results using IR, CD, Raman and NMR spectroscopies, and reasons for the variations in interpretation are discussed.     

Reassessment of the random coil conformation: vibrational CD study of proline oligopeptides and related polypeptides.

The "random coil" conformational problem is examined by comparison of vibrational CD (VCD) spectra of various polypeptide model systems with that of proline oligomers [(Pro)n] and poly(L-proline). VCD, ir and uv CD spectra of blocked L-proline oligopeptides [(Pro)n, n = 2-12] in different solvents are reported and compared to the spectra of poly(L-proline) II, poly(L-glutamic acid), and unblocked proline oligomers. Based on the chain-length dependence of the VCD and electronic CD (ECD) spectra of proline oligomers, it is established that VCD spectra are dominated by short-range interactions. The VCD of random coil model polypeptides is shown to be identical in shape but smaller in magnitude than poly(L-proline) II and of similar magnitude to that of (Pro)n (n = 3, 4). Based on the spectral evidence, it is concluded that the "random coil" conformation has a large fraction of helical regions, conformationally similar to the left-handed, 3(1) polyproline II helix, as was previously suggested by Krimm and co-workers. This conclusion is further supported by studies of effects of salt (CaCl2, LiBr, LiClO4), temperature (5-75 degrees C), and pH on the VCD spectra of L-proline oligomers, poly(L-proline) II, and poly(L-glutamic acid). These show that, after each of these perturbations, a significant local ordering remains in the oligomers and polymers studied, and that charged polypeptides such as poly(L-glutamic acid) are more flexible than are polyproline or even L-proline oligomers.     

Optical spectroscopic elucidation of beta-turns in disulfide bridged cyclic tetrapeptides

Vibrational circular dichroism (VCD) spectroscopic features of type II beta-turns were characterized previously, but, criteria for differentiation between beta-turn types had not been established yet. Model tetrapeptides, cyclized through a disulfide bridge, were designed on the basis of previous experimental results and the observed incidence of amino acid residues in the i + 1 and i + 2 positions in beta-turns, to determine the features of VCD spectra of type I and II beta-turns. The results were correlated with electronic circular dichroism (ECD) spectra and VCD spectra calculated from conformational data obtained by molecular dynamics (MD) simulations. All cyclic tetrapeptides yielded VCD signals with a higher frequency negative and a lower frequency positive couplet with negative lobes overlapping. MD simulations confirmed the conformational homogeneity of these peptides in solution. Comparison with ECD spectroscopy, MD, and quantum chemical calculation results suggested that the low frequency component of VCD spectra originating from the tertiary amide vibrations could be used to distinguish between types of beta-turn structures. On the basis of this observation, VCD spectroscopic features of type II and VIII beta-turns and ECD spectroscopic properties of a type VIII beta-turn were suggested. The need for independent experimental as well as theoretical investigations to obtain decisive conformational information was recognized.     

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.     

Noncovalent interactions of peptides with porphyrins in aqueous solution: conformational study using vibrational CD spectroscopy.

Noncovalent interactions of poly(L-lysine) (PL), oligopeptides L-lysyl-L-alanyl-L-alanine and (L-lysyl-L-alanyl-L-alanine)(2) with meso-tetrakis(4-sulfonatophenyl)porphine (TPPS), and poly(L-glutamic acid) (PLGA) with meso-tetrakis(1-methyl-4-pyridyl)porphine tetra-p-tosylate (TMPyP) in aqueous solutions have been studied using combination of spectroscopic methods: Vibrational circular dichroism (VCD) spectroscopy in the mid-infrared region provides a direct information on conformational changes of the polypeptides and oligopeptides caused by interactions with porphyrins; ultraviolet-visible absorption, fluorescence, and electronic circular dichroism (ECD) reveal the aggregation characterization of the porphyrin part of the complexes. Interactions of TPPS with tripeptide, hexapeptide, and PL containing about ten amino acid residues in the molecular chain are accompanied with the changes of VCD patterns in the amide I' region. In these cases, the conformation of the oligopeptide part of complexes is obviously influenced by interactions with TPPS and partial changes of random coil structure are observed in VCD. When PL was composed of the hundreds of lysine residues, just a weak intensity decrease was detected and the shape of VCD spectrum typical for the random coil structure was preserved. As follows from the uv-vis absorption and fluorescence spectra, porphyrin molecules are attached to peptides by electrostatic interaction as a monomer or dimer and interaction between porphyrin and peptide depends on the polypeptide chain length. For the PLGA-TMPyP system with PLGA containing from tens to hundreds of glutamic acid residues in the chain, the VCD spectra were unchanged when TMPyP was presented in the aqueous solution of PLGA and random coil conformation of PLGA-TMPyP aggregates was preserved.     

Vibrational circular dichroism of polypeptides. II. Solution amide II and deuteration results

Vibrational CD (VCD) of amide I and II vibrations of several α-helical polypeptides have been measured in solution. For the amide II as well as the amide I [previously published: Lal, B.B. & Nafie, L.A. (1982) Biopolymers 21 , 2161] we find the VCD to be characteristic of the polypeptide secondary structure. Amide II bands of right-handed α helices were all found to have negative VCD and to have their maximum rotational strength for the parallel (low-energy) component. However, left-handed α helices formed from L -amino acids gave positive amide II bands at higher frequencies than found for the right-handed helices, indicating that the VCD was sensitive to the stereochemical difference. The amide-I VCD spectra of some deuterated right-handed α-helical polypeptides have a new negative feature to low frequency that does not reflect theoretical predictions but also appears to be stereochemically sensitive. Amide-II and amide-A VCD of a few deuterated polypeptides imply retention of the secondary-structure-dependent characteristics seen in the hydrogenated VCD.     

Vibrational circular dichroism of A-, B-, and Z-form nucleic acids in the PO2-stretching region.

Vibrational circular dichroism (VCD) spectra were measured for H2O solutions of several natural and model DNAs (single and double strands, oligomers and polymers) in the B-form, poly(dG-dC)-poly(dG-dC) in the Z-form, and various duplex RNAs in an A-form over the PO2-stretching region. Only the symmetric PO2 stretch at approximately 1075 cm-1 yields a significant intensity VCD signal. Differences of the PO2-stretching VCD spectra found for these conformational types are consistent with the spectral changes seen in the base region, but no sequence dependence was seen in contrast to VCD for base modes. The B to Z transition is accompanied by an inversion of the PO2- VCD spectra, which is characteristic of the change in the helical sense of the nucleic acid backbone. A-RNAs give rise to the same sense of couplet VCD as do B-DNAs but have a somewhat different shape because of overlapping ribose modes. These PO2- VCD spectral characteristics have been successfully modeled using simple dipole coupling calculations. The invariability of the symmetric PO2- stretching mode VCD spectra to the base sequence as opposed to that found for the C = O stretching and base deformation modes is evidence that this mode will provide a stable indication of the DNA helical sense.     

VCD spectroscopic and molecular dynamics analysis of the Trp-cage miniprotein TC5b

TC5b is a 20 residue polypeptide notable for its compact tertiary structure, a rarity for a short peptide. This structure is due to the "Trp-cage" motif, an association of aromatic, Pro, and Gly residues. The structure of TC5b has been fully characterized by NMR and electronic circular dichroism (ECD) studies, but has never been studied with vibrational circular dichroism (VCD) spectroscopy, which may reveal finer structure. In this study, we examine the VCD spectra of TC5b to characterize the spectroscopic signature of the peptide and its comprising structural elements. TC5b exhibited a negative-positive-negative triplet which is associated with alpha-helical structure in deuterated solvents but also signs of a polyproline II (PPII) helix in the amide I' region. Detection of this element was complicated by the aforementioned triplet form, as well as by an upfrequency shift in PPII helical elements due to the use of the deuterated organic solvents DMSO-d(6) and TFE-d(1). Nevertheless, while ECD spectra showed only alpha-helical structure for TC5b, VCD spectroscopy revealed a more complex structure which was in agreement with NMR results. VCD spectroscopy also showed a rapid conformational change of the peptide at temperatures above 35 degrees C in D(2)O and in aqueous solvent with greater than 75% DMSO-d(6) content. Molecular dynamics (MD) simulations to investigate this latter effect of DMSO-d(6) on TC5b were conducted in DMSO and 50% (v/v) DMSO in H(2)O. In DMSO unfolding of the peptide was rapid while in 50% (v/v) DMSO in H(2)O the unfolding was more gradual.     

Helical nature of poly(dI-dC).poly(dI-dC). Vibrational circular dichroism results.

Poly(dI-dC).poly(dI-dC) was studied using vibrational circular dichroism and IR spectroscopy in both the base deformation C = O and symmetric PO2- stretching regions. VCD spectra of this duplex under low salt conditions are consistent with its having a B-form structure. Addition of 5 M NaCl leads to relatively uniform VCD intensity loss which is consistent with loss of helical structure rather than formation of an intermediate state between the B and Z forms. This duplex polymer under high salt conditions with added NiCl2 shows aggregation effects, but its IR and VCD spectra have characteristic features of the Z-form DNA conformation. The cooperative change of backbone and base pair structure upon thermal denaturation is indicated by the simultaneous collapse of the VCD at 65 degrees C in both the PO2- and C = O stretching regions. This study further demonstrates that the VCD bandshape of a specific localized nucleic acid vibrational transition can be a useful indicator of the helical handedness. The empirical conformational interpretations are supported by simulated VCD spectra, which are in excellent agreement with the experimental results, based on dipole coupling calculations.     

Enhanced sensitivity to conformation in various proteins. Vibrational circular dichroism results

Vibrational circular dichroism (VCD) spectra of several globular proteins dissolved in D2O are presented and compared to conventional UV-CD results. It can be seen that, for the alpha, beta, and alpha + beta categories of Levitt and Chothia [(1976) Nature 261, 552], VCD evidences much larger band shape variations, including sign alteration, than does UV-CD. A direct parallel is seen between the VCD of the alpha-helix found in model polypeptides and the amide I' VCD of myoglobin. Since all structural aspects of the protein contribute to the VCD on a roughly equal footing, a similar correlation of the chymotrypsin amide I' VCD with that of beta-sheet models is not as clear. In addition, the VCD of "random-coil"-type proteins is found to be clearly related to VCD results from "random-coil" polypeptides. Finally, simulations are presented to postulate the expected VCD for protein structures having conformations that lie between the limiting cases discussed here.     

Frequency analysis of infrared absorption and vibrational circular dichroism of proteins in D2O solution.

The IR absorption frequencies as derived from second derivatives of the Fourier transform IR spectra of the amide I' bands of globular proteins in D2O are compared to those obtained from band fitting of the vibrational circular dichroism (VCD) spectra. The two sets of frequencies are in very good agreement, yielding consistent ranges where amide I' VCD and IR features occur. Use of VCD to complement the IR allows one to add sign information to the frequency information so that features occurring in the overlapping frequency ranges that might arise from different secondary structures can be better discriminated. From this comparison, it is clear that correlation just of the frequency of a given IR transition to secondary structure can lead to a nonunique solution. Different sign patterns were identified for correlated groups of globular proteins in restricted frequency ranges that have been previously assigned to defined secondary structural elements. Hence, different secondary structural elements must contribute band components to a given frequency range.     

Origin of enhanced VCD in amyloid fibril spectra: Effect of deuteriation and pH

Supramolecular chirality of amyloid fibrils, protein aggregates related to many neurodegenerative diseases, is a remarkable property associated with fibril structure and polymorphism. Since its discovery almost 10 years ago there is still little understanding of this phenomenon, including the cause of the highly enhanced vibrational circular dichroism (VCD) intensity arising from fibril supramolecular chirality. In this study, VCD spectra, enhanced by filament supramolecular chirality, are presented for lysozyme and insulin fibrils above and below pH 2 and after deuterium exchange, above and below pD 2. Supramolecular chirality (observed by VCD) and fibril morphology (documented by atomic force microscopy) are not affected by protein deuteriation. In D₂O the fibril VCD sign pattern changes to fewer bands, with implications for the amide I/II origin of enhanced VCD intensity. Separation of amide I and II signals will facilitate calculations of enhanced VCD spectra of amyloid fibrils and enable a better understanding of the origin of the VCD sign pattern.     

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.     

Discriminating 3(10)- from alpha-helices: vibrational and electronic CD and IR absorption study of related Aib-containing oligopeptides

Model peptides based on -(Aib-Ala)(n)-, and (Aib)(n)-Leu-(Aib)(2) sequences, which have varying amounts of 3(10)-helical character, were studied by use of vibrational and electronic circular dichroism (VCD and ECD) and Fourier transform infrared (FTIR) absorption spectroscopies to test the correlation of spectral response and conformation. The data indicate that these peptides, starting from a length of about four to six residues, predominantly adopt a 3(10)-helical conformation at room temperature. The longest model peptides, depending on the series, may evidence some alpha-helical contribution to the spectra, while the shorter ones, with less than six residues, have much less order. The IR absorption spectra (as supported by theory) showed only small frequency changes between 3(10)- and alpha-helices. By contrast, solvent effects are a source of much bigger perturbations. The ECD results show that the intensity ratio for the approximately 222-nm to approximately 208-nm bands, while useful for distinguishing between these two helical types in some sequences, may have a narrower range of application than VCD. However, the VCD data presented here continue to support the proposed discrimination between alpha- and 3(10)-helices based on qualitative amide I and II bandshape differences. The present study shows the intensities of the 3(10)-helical amide I (peak-to-peak) to its amide II VCD to be of the same order and useful for discriminating them from alpha-helices, whose amide I dominates the amide II in intensity. This qualitative result is experimentally independent of the amount of alphaMe-substituted residues in the sequence. These experimental VCD results are consistent in detail with theoretical spectral simulations for Ac-(Ala)(8)-NH(2), Ac-(Aib-Ala)(4)-NH(2), and Ac-(Aib)(8)-NH(2) in 3(10)- and alpha-helical conformations.     

Quantitative IR spectrophotometry of peptide compounds in water (H2O) solutions. II. Amide absorption bands of polypeptides and fibrous proteins in alpha-, beta-, and random coil conformations.

Infrared spectra of poly(D,L-alanine), poly(L-glutamic acid), poly(L-lysine), silk fibroin, and tropomyosin have been registered for various conformations of the polypeptide chain. Assuming additivity of the main- and side-chain absorption, spectral parameters of amide I and II absorption bands corresponding to alpha-, beta-, and random coil conformations have been derived. The amide I band parameters for H2O and D2O have been compared.     

Structural transition during thermal denaturation of collagen in the solution and film states

Temperature dependent vibrational circular dichroism (VCD) spectra of type I collagen, in solution and film states, have been measured. These spectra obtained for solution sample suggest that the thermal denaturation of collagen results in transition from poly-L-proline II (PPII) to unordered structure. The PPII structure of collagen is identified by the presence of negative VCD couplet in the amide I region, while the formation of unordered structure is indicated by the disappearance of VCD in the amide I region. The temperature dependent spectra obtained for the supported collagen film indicated a biphasic transition, which is believed to be the first vibrational spectroscopic report to support a biphasic transition during thermal denaturation of collagen film. The temperature dependent spectra of collagen films suggest that the thermal stability of collagen structure depends on its state and decreases in the order: supported film > free standing film > solution state. These observations are believed to be significant in the VCD spectroscopic analysis of secondary structures of proteins and peptides.