Rina K. Dukor

Vibrational circular dichroism spectroscopy of selected oligopeptide conformations

Vibrational circular dichroism (VCD) has been shown to be a useful technique for characterization of the qualitative secondary structure type for linear polypeptides and oligopeptides. A brief review of characteristic spectral responses and applications is given. Since VCD is dependent on relatively short range interactions, it detects residual structure in such oligomers even if long range order is lost. VCD studies presented here for Lys oligomers as well as Lys and Glu polymers as a function of length, salt added and temperature, confirm residual local order in these random coils. Comparison to results with Pro oligomers, supports an interpretation that these extended structures have a left handed twist conformation. The coil VCD is shown to be significantly reduced in intensity by temperature increase and by decrease in peptide length. By contrast, for partially alpha-helical Ac-(AAKAA)3GY-NH2 oligomers, the spectrum changes to the high temperature Lys(n) shape on heating, first losing then gaining intensity, indicating an equilibrium shift between structured states, from helix to coil (locally ordered) forms. VCD is shown to be a useful technique for monitoring local order in otherwise random coil structures.

Mutarotation studies of poly-L-proline using FTIR, electronic and vibrational circular dichroism

FTIR, electronic and vibrational circular dichroism (ECD and VCD) are used to follow the mutarotation of poly-L-proline I (PLP I) to poly-L-proline II (PLP II) in D2O solution. While this process is well known, these studies elucidate the nature of the characteristic spectra of each form in an aqueous environment, which can be useful for qualitative structural diagnoses of proteins and peptides using these spectroscopic techniques. Further, our data demonstrate the presence of an intermediate in the transformation, at least in highly concentrated aqueous solution, which is characterized by an absorption band growing in at 1653 cm−1 and then decaying away as the transformation proceeds. Based on correlations to FTIR and VCD studies on L-proline and D,L alternate proline oligopeptides previously carried out in our laboratories, this intermediate structure can be assigned to there being a distribution of cis-trans linkages in the polymer. This contrasts with the cooperative conformational transition from PLP I to PLP II, proceeding sequentially from end to end, that has been proposed for this mutarotation in previous studies, primarily in different environments. Our results are consistent with early interpretations of hydrodynamic data. Related studies of collagen and poly-L-hydroxyproline indicate that both yield VCD spectra similar in shape to that of PLP II, as expected, and demonstrate that intra-chain, near neighbor interactions dominate the VCD.

Empirical studies of protein secondary structure by vibrational circular dichroism and related techniques. α-Lactalbumin and lysozyme as examples

Vibrational circular dichroism (VCD) has been shown to be sensitive to secondary structure in proteins and peptides and has been used as the basis for quantitative secondary-structure-prediction algorithms. However, the accuracy of these algorithms is not matched by the apparent qualitative sensitivity of the VCD spectra. This report provides examples of the use of VCD to follow structural change spectrally and to clarify the qualitative nature of the structural changes underlying the spectral variation. The VCD spectra and the complementary UV electronic CD (ECD) and FTIR spectra of alpha-lactalbumin (LA) have been studied as a function of pH, denaturation, Ca2+ ion and solvent conditions for several species. Spectral data for lysozyme were compared with those of LA because of their very similar crystal structures. In fact, these proteins in D2O-based pH 7 solution have quite different spectra using these optical techniques. Even for the LA proteins, the human differs from the bovine and goat species. Furthermore, under low pH conditions, where the LAs are in a reversibly denatured, molten globule form, the spectra are more similar, species variation is minimal and the spectral differences from lysozyme are in fact smaller. Our data are consistent with native, pH 7, alpha-lactalbumin having a less well organized structure than lysozyme, possibly in a dynamic sense. Conversely, in the low-pH, molten globule form of LA, tertiary structure is lost which could relax constraints that might distort the helical segments in the native form. The differences between the interpretation of our results and those from X-ray and NMR data may be due to motional sampling of various geometries in LA which all contribute to the spectral signatures seen in optical spectra but whose contributions are washed out in NMR or frozen out in the crystal structure. Part of this flexibility may relate to the rather large 3(10)-helical content in the LA protein structure. Fluctionality may have specific functional effects, perhaps allowing LA to bind better to beta-galactosyl transferase and form the biologically active lactose synthetase complex.

Vibrational circular dichroism studies of epidermal growth factor and basic fibroblast growth factor

Vibrational circular dichroism (VCD) studies are reported for two unrelated recombinant growth factor proteins: epidermal growth factor and basic fibroblast growth factor (bFGF). NMR, electronic CD, and bFGF X-ray studies indicate that these two proteins are primarily composed of beta-sheet and loop secondary structure elements with no detectable alpha-helices. Two reports on solution conformation of these proteins using FTIR absorption spectroscopy with subsequent resolution enhancement confirmed the presence of a large fraction of a beta-sheet conformation but in addition indicated the presence of large absorption bands in the 1650-1656 cm-1 region, which are typically assigned to alpha-helices. The VCD spectra of both proteins have band shapes that strongly resemble those of other high beta-sheet fraction proteins, such as the trypsin family of proteins. Quantitative analysis of the VCD spectra also indicates that these proteins are predominantly in beta-sheet and extended (other) conformations with very little alpha-helix fraction. These results agree with the CD interpretation and affirm that the FTIR peaks in the region 1650-1656 cm-1 can be assigned to loops. This study provides an example of the limitations of using FTIR frequencies alone for examination of protein secondary structure.

Biopolymer Conformational Studies With Vibrational Circular Dichroism

Vibrational Circular Dichroism (VCD) has proven to be useful for detecting conformational change and for characterizing secondary structures in polypeptides, proteins and nucleic acids. Recent progress in these areas is reviewed with an emphasis on the contribution of VCD studies to furthering the understanding of conformational aspects of these problems.

Techniques And Application Of FTIR Vibrational Circular Dichroism

Vibrational Circular Dichroism (VCD) has proven to be useful for detecting conformational change and for characterizing secondary structures for a variety of biopolymers. Use of FTIR-based VCD for such problems has been made possible with recent improvements in optical design and S/N. FTIR-VCD and dispersive VCD each have strengths that are optimal for specific problems. A comparison of techniques is presented in this lecture, and selected examples of VCD applications to polypeptides and proteins are given.