Conformation of unfolded and partially folded peptides and proteins probed by optical spectroscopy

Conformational plasticity in biomolecules gives rise to unique characteristics. How proteins fold into native three-dimensional structures has been a long-investigated mystery. One piece of information, i.e. intrinsic conformational propensities of individual amino acids in a polypeptide chain, encodes the folding energy landscape of a protein, which facilitates proteins to fold spontaneously, without randomly sampling the ensemble of accessible conformations. The fact that cytochrome c undergoes conformational changes in biological processes underscores the importance of heterogeneity in biomolecules.;We have determined the conformational ensembles sampled by amino acids. Our results indicate that amino acids with aliphatic side chains substantially sample polyproline II (PPII) conformations, as well as a region between PPII and beta-regions. Charged residues, E, K and R behave like aliphatic residues, while D samples regions whose dihedral angles are similar to those found in turn structures. A similar sampling of dihedral angles found in turn motifs was found for polar residues as well.;We have characterized intermediate states adopted in thermal and alkaline unfolding of ferricytochrome c. We demonstrated that states IIIh and IV are different, contrary to the current belief. Most importantly, we have shown the need for a comprehensive thermodynamic analysis of all adopted states of ferricytochrome c under well-defined conditions which would consider that not only the "ground state" but also thermally excited and (partially) unfolded states are pH dependent.;Finally, we have estimated deformations of the heme groups in three ferrocytochrome c isoforms; horse, chicken and yeast. We obtained that ruffling was the largest deformation experienced by all investigated hemes with chicken being the most ruffled followed by horse and yeast. The heme group in horse was the most saddled followed by yeast, then chicken. We determined that the heme of chicken experienced the most doming followed by horse and yeast. Finally, the heme group of horse was the most propellered. The main saddling and ruffling deformations from crystal and MD structures correlate with our results, whereas MD simulations better account for smaller deformations (doming and propellering), due to the uncertainty of crystal structure coordinates relating to errors in small deformations.