Inherent chirality dominates the visible/near-ultraviolet CD spectrum of rhodopsin.

The visible (alpha) and near-UV (beta) CD bands of rhodopsin have been studied extensively, but their source(s) have never been definitively established. Do they result from the intrinsic chirality of the polyene chromophore of the protonated Schiff base of retinal (retPSB) or from the coupling of the transitions of this chromophore with those of protein groups? We have calculated the contributions of these two mechanisms to the CD of rhodopsin. The intrinsic CD of the retPSB chromophore was calculated using time-dependent density functional theory (TDDFT) and, for comparison, the semiempirical ZINDO method. First-order perturbation theory was used to calculate the effects of coupling of the retPSB transitions with the pi pi* transitions of the aromatic chromophores and the pi pi* and n pi* transitions of the peptide groups in rhodopsin. Calculations were performed for eight structures based upon the two molecules in the asymmetric unit of four crystal structures. The most reliable results were obtained from TDDFT calculations on the structure of Okada et al. (J. Mol. Biol. 2004, 342, 571), PDB 1U19. Averaging over the two molecules in the asymmetric unit, the intrinsic rotational strengths are 0.62 +/- 0.00 DBM (Debye-Bohr magneton) and 0.90 +/- 0.03 DBM for the alpha- and beta-bands, respectively. The contributions from coupling with protein groups are, respectively, -0.32 +/- 0.05 and -0.01 +/- 0.03 DBM. Our results show that the visible/near-UV CD bands of rhodopsin are determined by the intrinsic chirality of the retPSB chromophore and that the contributions of coupling with the protein are significantly smaller for the alpha-band and negligible for the beta-band.

Theoretical investigation of the photoinitiated folding of HP-36.

A computational model was developed to examine the phototriggered folding of a caged protein, a protein modified with an organic photolabile cross-linker. Molecular dynamics simulations of the modified 36-residue fragment of subdomain B of chicken villin head piece with a photolabile linker were performed, starting from both the caged and the uncaged structures. Construction of a free-energy landscape, based on principal components as well as on radius of gyration versus root-mean-square deviation, and circular dichroism calculations were employed to characterize folding behavior and structures. The folded structures observed in the molecular dynamics trajectories were found to be similar to that of the wild-type protein, in agreement with the published experimental results. The free-energy landscapes of the modified and wild-type proteins have similar topology, suggesting common thermodynamic/kinetic behavior. The existence of small differences in the free-energy surface of the modified protein from that of the native protein, however, indicates subtle differences in the folding behavior.

On the analysis of membrane protein circular dichroism spectra.

Analysis of circular dichroism spectra of proteins provides information about protein secondary structure. Analytical methods developed for such an analysis use structures and spectra of a set of reference proteins. The reference protein sets currently in use include soluble proteins with a wide range of secondary structures, and perform quite well in analyzing CD spectra of soluble proteins. The utility of soluble protein reference sets in analyzing membrane protein CD spectra, however, has been questioned in a recent study that found current reference protein sets to be inadequate for analyzing membrane proteins. We have examined the performance of reference protein sets available in the CDPro software package for analyzing CD spectra of 13 membrane proteins with available crystal structures. Our results indicate that the reference protein sets currently available for CD analysis perform reasonably well in analyzing membrane protein CD spectra, with performance indices comparable to those for soluble proteins. Soluble + membrane protein reference sets, which were constructed by combining membrane proteins with soluble protein reference sets, gave improved performance in both soluble and membrane protein CD analysis.

Structural composition of betaI- and betaII-proteins.

Circular dichroism spectra of proteins are sensitive to protein secondary structure. The CD spectra of alpha-rich proteins are similar to those of model alpha-helices, but beta-rich proteins exhibit CD spectra that are reminiscent of CD spectra of either model beta-sheets or unordered polypeptides. The existence of these two types of CD spectra for beta-rich proteins form the basis for their classification as betaI- and betaII-proteins. Although the conformation of beta-sheets is largely responsible for the CD spectra of betaI-proteins, the source of betaII-protein CD, which resembles that of unordered polypeptides, is not completely understood. The CD spectra of unordered polypeptides are similar to that of the poly(Pro)II helix, and the poly(Pro)II-type (P2) structure forms a significant fraction of the unordered conformation in globular proteins. We have compared the beta-sheet and P2 structure contents in beta-rich proteins to understand the origin of betaII-protein CD. We find that betaII-proteins have a ratio of P2 to beta-sheet content greater than 0.4, whereas for betaI-proteins this ratio is less than 0.4. The beta-sheet content in betaI-proteins is generally higher than that in betaII-proteins. The origin of two classes of CD spectra for beta-rich proteins appears to lie in their relative beta-sheet and P2 structure contents.

Heme distortions in sperm-whale carbonmonoxy myoglobin: correlations between rotational strengths and heme distortions in MD-generated structures.

We have investigated the effects of heme rotational isomerism in sperm-whale carbonmonoxymyoglobin using computational techniques. Several molecular dynamics simulations have been performed for the two rotational isomers A and B, which are related by a 180 degrees rotation around the alpha-gamma axis of the heme, of sperm-whale carbonmonoxy myoglobin in water. Both neutron diffraction and NMR structures were used as starting structures. In the absence of an experimental structure, the structure of isomer B was generated by rotating the heme in the structure of isomer A. Distortions of the heme from planarity were characterized by normal coordinate structural decomposition and by the angle of twist of the pyrrole rings from the heme plane. The heme distortions of the neutron diffraction structure were conserved in the MD trajectories, but in the NMR-based trajectories, where the heme distortions are less well defined, they differ from the original heme deformations. The protein matrix induced similar distortions on the hemes in orientations A and B. Our results suggest that the binding site prefers a particular macrocycle conformation, and a 180 degrees rotation of the heme does not significantly alter the protein's preference for this conformation. The intrinsic rotational strengths of the two Soret transitions, separated according to their polarization in the heme plane, show strong correlations with the ruffling deformation and the average twist angle of the pyrrole rings. The total rotational strength, which includes contributions from the chromophores in the protein, shows a weaker correlation with heme distortions.

Independent tyrosyl contributions to the CD of Ff gene 5 protein and the distinctive effects of Y41H and Y41F mutants on protein-protein cooperative interactions.

The gene 5 protein (g5p) of the Ff virus contains five Tyr, individual mutants of which have now all been characterized by CD spectroscopy. The protein has a dominant tyrosyl 229-nm L(a) CD band that is shown to be approximately the sum of the five individual Tyr contributions. Tyr41 is particularly important in contributing to the high cooperativity with which the g5p binds to ssDNA, and Y41F and Y41H mutants are known to differ in dimer-dimer packing interactions in crystal structures. We compared the solution structures and binding properties of the Y41F and Y41H mutants using CD spectroscopy. Secondary structures of the mutants were similar by CD analyses and close to those derived from the crystal structures. However, there were significant differences in the binding properties of the two mutant proteins. The Y41H protein had an especially low binding affinity and perturbed the spectrum of poly[d(A)] in 2 mM Na(+) much less than did Y41F and the wild-type gene 5 proteins. Moreover, a change in the Tyr 229 nm band, assigned to the perturbation of Tyr34 at the dimer-dimer interface, was absent in titrations with the Y41H mutant under low salt conditions. In contrast, titrations with the Y41H mutant in 50 mM Na(+) exhibited typical CD changes of both the nucleic acid and the Tyr 229-nm band. Thus, protein-protein and g5p-ssDNA interactions appeared to be mutually influenced by ionic strength, indicative of correlated changes in the ssDNA binding and cooperativity loops of the protein or of indirect structural constraints.