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

Andrew Michael Hagarman

doi:10.17918/etd-3313

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. In order to estimate intrinsic conformational propensities of individual amino acids we use a protocol that allows us to simulate experimental isotropic Raman, anisotropic Raman, FTIR and vibrational circular dichroism spectra and a set of six NMR J-coupling constants by using a superposition of statistically weighted two-dimensional Gaussian distributions representing sterically allowed regions of the Ramachandran space. We use the host-guest motif glycine-x-glycine, where x is confined to a set of amino acids representing aliphatic (A, V, L, M, I), aromatic (F, Y), charged (E, D, R, K) and polar (S, T, C, N) residues. The selection of glycine hosts was imperative to minimize nearest-neighbor effects that would modulate the conformational propensity of the central residue. 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. In order to estimate intrinsic conformational propensities of individual amino acids we use a protocol that allows us to simulate experimental isotropic Raman, anisotropic Raman, FTIR and vibrational circular dichroism spectra and a set of six NMR J-coupling constants by using a superposition of statistically weighted two-dimensional Gaussian distributions representing sterically allowed regions of the Ramachandran space. We use the host-guest motif glycine-x-glycine, where x is confined to a set of amino acids representing aliphatic (A, V, L, M, I), aromatic (F, Y), charged (E, D, R, K) and polar (S, T, C, N) residues. The selection of glycine hosts was imperative to minimize nearest-neighbor effects that would modulate the conformational propensity of the central residue. 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.

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

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

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