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

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.Ph.D., Physical Chemistry -- Drexel University, 201

Optical Band Splitting and Electronic Perturbations of the Heme Chromophore in Cytochrome c at Room Temperature Probed by Visible Electronic Circular Dichroism Spectroscopy

AbstractWe have measured the electronic circular dichroism (ECD) of the ferri- and ferro-states of several natural cytochrome c derivatives (horse heart, chicken, bovine, and yeast) and the Y67F mutant of yeast in the region between 300 and 750nm. Thus, we recorded the ECD of the B- and Q-band region as well as the charge-transfer band at ∼695nm. The B-band region of the ferri-state displays a nearly symmetric couplet at the B0-position that overlaps with a couplet 790cm−1 higher in energy, which we assigned to a vibronic side-band transition. For the ferro-state, the couplet is greatly reduced, but still detectable. The B-band region is dominated by a positive Cotton effect at energies lower than B0 that is attributed to a magnetically allowed iron→heme charge-transfer transition as earlier observed for nitrosyl myoglobin and hemoglobin. The Q-band region of the ferri-state is poorly resolved, but displays a pronounced positive signal at higher wavenumbers. This must result from a magnetically allowed transition, possibly from the methionine ligand to the dxy-hole of Fe3+. For the ferro-state, the spectra resolve the vibronic structure of the Qv-band. A more detailed spectral analysis reveals that the positively biased spectrum can be understood as a superposition of asymmetric couplets of split Q0 and Qv-states. Substantial qualitative and quantitative differences between the respective B-state and Q-state ECD spectra of yeast and horse heart cytochrome c can clearly be attributed to the reduced band splitting in the former, which results from a less heterogeneous internal electric field. Finally, we investigated the charge-transfer band at 695nm in the ferri-state spectrum and found that it is composed of at least three bands, which are assignable to different taxonomic substates. The respective subbands differ somewhat with respect to their Kuhn anisotropy ratio and their intensity ratios are different for horse and yeast cytochrome c. Our data therefore suggests different substate populations for these proteins, which is most likely assignable to a structural heterogeneity of the distal Fe-M80 coordination of the heme chromophore

Asymmetric band profile of the Soret band of deoxymyoglobin is caused by electronic and vibronic perturbations of the heme group rather than by a doming deformation

Journal of Chemical Physics, 127(13): pp. 135103We measured the Soret band of deoxymyoglobin deoxyMb , myoglobin cyanide MbCN , and aquo-metmyoglobin all from horse heart with absorption and circular dichroism CD spectroscopies. A clear non-coincidence was observed between the absorption and CD profiles of deoxyMb and MbCN, with the CD profiles red- and blueshifted with respect to the absorption band position, respectively. On the contrary, the CD and absorption profiles of aquametMb were nearly identical. The observed noncoincidence indicates a splitting of the excited B state due to heme-protein interactions. CD and absorption profiles of deoxyMb and MbCN were self-consistently analyzed by employing a perturbation approach for weak vibronic coupling as well as the relative intensities and depolarization ratios of seven bands in the respective resonance Raman spectra measured with B-band excitation. The respective By component was found to dominate the observed Cotton effect of both myoglobin derivatives. The different signs of the noncoincidences between CD and absorption bands observed for deoxyMb and MbCN are due to different signs of the respective matrix elements of A1g electronic interstate coupling, which reflects an imbalance of Gouterman’s 50:50 states. The splitting of the B band reflects contributions from electronic and vibronic perturbations of B1g symmetry. The results of our analysis suggest that the broad and asymmetric absorption band of deoxyMb results from this band splitting rather than from its dependence on heme doming. Thus, we are able to explain recent findings that the temperature dependences of CO rebinding to myoglobin and the Soret band profile are uncorrelated Ormos et al., Proc. Natl. Acad. Sci U.S.A. 95, 6762 1998

Cu(II) and Ni(II) Interactions with the Terminally Blocked Hexapeptide Ac-Leu-Ala-His-Tyr-Asn-Lys-amide Model of Histone H2B (80–85)

The N- and C-terminal blocked hexapeptide Ac-Leu-Ala-His-Tyr-Asn-Lys-amide (LAHYNK) representing the 80–85 fragment of histone H2B was synthesized and its interactions with Cu(II) and Ni(II) ions were studied by potentiometric, UV-Vis, CD, EPR, and NMR spectroscopic techniques in solution. Our data reveal that the imidazole N(3) nitrogen atom is the primary ligating group for both metal ions. Sequential amide groups deprotonation and subsequent coordination to metal ions indicated an {Nimidazole, 3Namide} coordination mode above pH∼9, in all cases. In the case of Cu(II)-peptide system, the almost exclusive formation of the predominant species CuL in neutral media accounting for almost 98% of the total metal ion concentration at pH 7.3 strongly indicates that at physiological pH values the sequence -LAHYNK- of histone H2B provides very efficient binding sites for metal ions. The imidazole pyrrole N(1) ionization (but not coordination) was also detected in species CuH−4L present in solution above pH ∼ 11