Article

Solvent dependence of PII conformation in model alanine peptides

Department of Biochemistry and Molecular Biology, Colorado State University, Fort Collins, Colorado, United States
Journal of the American Chemical Society (Impact Factor: 11.44). 12/2004; 126(46):15141-50. DOI: 10.1021/ja047594g
Source: PubMed

ABSTRACT Alanine residues in two model peptides, the pentapeptide AcGGAGGNH(2) and the 11mer AcO(2)A(7)O(2)NH(2), have been reported to have substantial PII conformation in water. The PII structure in both peptides is sensitive to solvent. In the presence of the organic solvent TFE, the conformation of the pentamer changes from PII to internally H-bonded gamma or beta turns, while the chain with seven alanines forms alpha helix. The PII structure in the 11mer is more stable than that in the shorter peptide as the TFE concentration increases. For the pentamer, a comparison of short-chain aliphatic alcohols to water shows that the PII content decreases in the order water > methanol > ethanol > 2-propanol, linearly according to empirical scales of solvent polarity. Thus, depending on the extent of local solvation as folding progresses, the peptide backbone as modeled by alanine oligomers shifts from PII to internally H-bonded (gamma or beta turn) conformations and to alpha helix in longer segments. On the other hand, the PII content of AcO(2)A(7)O(2)NH(2) increases significantly in the presence of guanidine, as does that of oligoproline peptides, while detergent sodium dodecyl sulfate (SDS) favors alpha helix in this peptide. The shorter peptide does not show a parallel increase in PII with guanidine.

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We have thus confirmed alanine’s high propensity to adopt dihedral angles in the PPII distribution and determined that aliphatic and positively charged residues Conformational plasticity in biomolecules gives rise to unique characteristics. How a protein folds into its native three-dimensional structure has been a long investigated mystery, but it is tied into conformational sampling of polymeric chains of amino acids. One critical piece of information, i.e. intrinsic conformational propensities of individual amino acids in a polypeptide chain, encodes the folding energy landscape of a protein. This funneled landscape facilitates the ability for proteins to fold spontaneously, without randomly sampling the ensemble of accessible conformations. Also, the fact that an essential protein in the electron transport chain, cytochrome c, undergoes conformational changes in many biological processes underscores the importance of conformational heterogeneity in biomolecules. 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We have thus confirmed alanine’s high propensity to adopt dihedral angles in the PPII distribution and determined that aliphatic and positively charged residues extent of band splitting caused by electrostatic interactions between the heme group and the protein was determined by a vibronic analysis of the B-band ECD and absorption spectra. We demonstrated that the states IIIh and IV are thermodynamically and also conformationally different, contrary to the current belief. With respect to ferricytochrome c our results suggest that the overall structure is maintained in the intermediate state populated above 323 K. Conformational changes might involve increasing distances between the heme and aromatic residues such as F82 and a reduced nonplanarity of the heme macrocycle. The band splitting is substantially reduced in the unfolded states, but the heme environment encompassing H18 and the two cysteine residues 14 and 17 is most likely still intact and covalently bound to the heme chromophore. Most importantly, we have shown the need for a comprehensive thermodynamic analysis of all native and non-native states of ferricytochrome c under well-defined conditions which would explicitly consider the fact that not only the “ground state” populated at room temperature but also the thermally excited, partially or mostly unfolded states are still pH dependent. Cytochrome c is in a class of proteins with high redox potentials. Its comparatively high redox potential is stabilized by a hexacoordinated central iron atom in the heme c which is coordinated to a sulfur of a methionine in the surrounding protein matrix at the distal coordination site, as well as by interactions with the internal electric field created by ionizable groups within the heme pocket. Thus, deformations of the heme group are functionally relevant in modulating the redox potential. We have used polarized resonance Raman spectroscopy to exploit the depolarization ratios and normalized extent of band splitting caused by electrostatic interactions between the heme group and the protein was determined by a vibronic analysis of the B-band ECD and absorption spectra. We demonstrated that the states IIIh and IV are thermodynamically and also conformationally different, contrary to the current belief. With respect to ferricytochrome c our results suggest that the overall structure is maintained in the intermediate state populated above 323 K. Conformational changes might involve increasing distances between the heme and aromatic residues such as F82 and a reduced nonplanarity of the heme macrocycle. The band splitting is substantially reduced in the unfolded states, but the heme environment encompassing H18 and the two cysteine residues 14 and 17 is most likely still intact and covalently bound to the heme chromophore. Most importantly, we have shown the need for a comprehensive thermodynamic analysis of all native and non-native states of ferricytochrome c under well-defined conditions which would explicitly consider the fact that not only the “ground state” populated at room temperature but also the thermally excited, partially or mostly unfolded states are still pH dependent. Cytochrome c is in a class of proteins with high redox potentials. Its comparatively high redox potential is stabilized by a hexacoordinated central iron atom in the heme c which is coordinated to a sulfur of a methionine in the surrounding protein matrix at the distal coordination site, as well as by interactions with the internal electric field created by ionizable groups within the heme pocket. Thus, deformations of the heme group are functionally relevant in modulating the redox potential. We have used polarized resonance Raman spectroscopy to exploit the depolarization ratios and normalized intensities of Raman active bands in the low frequency Soret excited Raman spectrum for an estimation of planar and non-planar deformations of the heme active sites in three different reduced cytochrome c isoforms; horse, chicken and a mutated – to avoid aggregation - Saccromyces Cerevisae (yeast). We thus obtained that ruffling was the largest deformation experienced by all investigated hemes with chicken being the most ruffled folloed by horse heart and yeast. Concerning the saddling deformations, the heme group in horse heart was the most followed by yeast, then chicken. We determined that the heme c of chicken experienced the most doming followed by horse heart and yeast. Finally, the heme group of horse heart was determined to be the most propellered. The main saddling and ruffling deformations from crystal and MD structures compare well with our results, whereas MD simulations better account for smaller deformations like doming and propellering, due to the fact that the uncertainty of crystal structures coordinates relates to high error in small deformations.
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