Narasimha Sreerama

Computation and analysis of protein circular dichroism spectra.

Publisher Summary This chapter presents computation and analysis of protein circular dichroism (CD) spectra. The origins of electronic CD in proteins, theoretical methods for computing protein CD, and empirical analysis of CD for estimating structural composition of proteins are described. The phenomenon of CD involves the absorption of light and it can be considered as a special type of absorption spectroscopy. The CD spectra of proteins are generally divided into three wavelength ranges, based on the energy of the electronic transitions that dominate in the given range. The basic approach used to compute the CD of complex systems, such as proteins and nucleic acids, is the divide and conquer approach. In addition to the isotropic atomic polarizabilities, the anisotropic polarizability of the first electrically allowed peptide transition was included in the dipole interaction model for protein CD calculations. Polarizability anisotropy data for simple amides were used for obtaining polarizability parameters. It is found that the CD spectrum of the typical β sheet has a negative band near 215-nm and a positive band near 198 nm.

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 βI‐ and βII‐proteins

Circular dichroism spectra of proteins are sensitive to protein secondary structure. The CD spectra of α‐rich proteins are similar to those of model α‐helices, but β‐rich proteins exhibit CD spectra that are reminiscent of CD spectra of either model β‐sheets or unordered polypeptides. The existence of these two types of CD spectra for β‐rich proteins form the basis for their classification as βI‐ and βII‐proteins. Although the conformation of β‐sheets is largely responsible for the CD spectra of βI‐proteins, the source of βII‐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 β‐sheet and P2 structure contents in β‐rich proteins to understand the origin of βII‐protein CD. We find that βII‐proteins have a ratio of P2 to β‐sheet content greater than 0.4, whereas for βI‐proteins this ratio is less than 0.4. The β‐sheet content in βI‐proteins is generally higher than that in βII‐proteins. The origin of two classes of CD spectra for β‐rich proteins appears to lie in their relative β‐sheet and P2 structure contents.

Molecular dynamics simulations of polypeptide conformations in water: A comparison of α, β, and poly(pro)II conformations

A significant fraction of the so‐called “random coil” residues in globular proteins exists in the left‐handed poly(Pro)II conformation. In order to compare the behavior of this secondary structure with that of the other regular secondary structures, molecular dynamics simulations, with the GROMOS suite of programs, of an alanine octapeptide in water, in α‐helix, β‐strand, and left‐handed poly(Pro)II conformations, have been performed. Our results indicate a limited flexibility for the α‐helix conformation and a relatively larger flexibility for the β‐strand and poly(Pro)II conformations. The behavior of oligopeptides with a starting configuration of β‐strand and poly(Pro)II conformations, both lacking interchain hydrogen bonds, were similar. The (ϕ, ψ) angles reflect a continuum of structures including both β and PII conformations, but with a preference for local PII regions. Differences in the network of water molecules involved in hydrogen bonding with the backbone of the polypeptide were observed in local regions of β and PII conformations. Such water bridges help stabilize the PII conformation relative to the β conformation. Proteins 1999;36:400–406.

Comment on “Improving protein circular dichroism calculations in the far-ultraviolet through reparametrizing the amide chromophore” [J. Chem. Phys. 109, 782 (1998)]

Circular dichroism spectra for 23 proteins have been calculated using transition parameters from experiments on model amides and semiempirical molecular orbital (MO) calculations. The results are substantially better than those reported by Hirst. The improvements result primarily from using distributed dipoles (monopoles), rather than the point dipoles used by Hirst.

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.

Thermal and urea-induced unfolding in T7 RNA polymerase: calorimetry, circular dichroism and fluorescence study.

Structural changes in T7 RNA polymerase (T7RNAP) induced by temperature and urea have been studied over a wide range of conditions to obtain information about the structural organization and the stability of the enzyme. T7RNAP is a large monomeric enzyme (99 kD). Calorimetric studies of the thermal transitions in T7RNAP show that the enzyme consists of three cooperative units that may be regarded as structural domains. Interactions between these structural domains and their stability strongly depend on solvent conditions. The unfolding of T7RNAP under different solvent conditions induces a highly stable intermediate state that lacks specific tertiary interactions, contains a significant amount of residual secondary structure, and undergoes further cooperative unfolding at high urea concentrations. Circular dichroism (CD) studies show that thermal unfolding leads to an intermediate state that has increased β‐sheet and reduced α‐helix content relative to the native state. Urea‐induced unfolding at 25°C reveals a two‐step process. The first transition centered near 3 M urea leads to a plateau from 3.5 to 5.0 M urea, followed by a second transition centered near 6.5 M urea. The CD spectrum of the enzyme in the plateau region, which is similar to that of the enzyme thermally unfolded in the absence of urea, shows little temperature dependence from 15° to 60°C. The second transition leads to a mixture of poly(Pro)II and unordered conformations. As the temperature increases, the ellipticity at 222 nm becomes more negative because of conversion of poly(Pro)II to the unordered conformation. Near‐ultraviolet CD spectra at 25°C at varying concentrations of urea are consistent with this picture. Both thermal and urea denaturation are irreversible, presumably because of processes that follow unfolding.

Characterization of proline-containing right-handed α-helix by molecular dynamics studies

Proline residues play a special role in shaping the secondary and tertiary structures of proteins. Many of these aspects have been studied in great detail. Current interest lies in elucidating the structure of right-handed alpha-helical fragments which contain proline in the middle of the helix. Such structures play an important role in membrane proteins and in the tight packing of globular proteins. Analysis of several crystal structures and energy minimization using flexible geometry have elucidated the nature of the bend produced by proline in the right-handed alpha-helical structure. Molecular dynamics (MD) simulation studies are ideally suited to characterize rigidity or flexibility in different parts of the molecule and can also give an idea of various conformations of the molecule which can exist at a given temperature. Hence, MD studies on Ace-(Ala)6-Pro-(Ala)3-NHMe have been carried out for 100 ps after equilibration and the resulting trajectories have been analyzed. Information regarding the average values, r.m.s. fluctuations of internal parameters and the time spent in different conformations are discussed. Energy minimization has been carried out on selected MD simulated points in order to analyze the characteristics of different conformations.

An ab-initio study of the proton affinity of conjugated schiff-base and related nitrogen compounds: an analysis of the triggering site of bacteriorhodopsin

The chemical groups which take part in the proton transfer reaction in bacteriorhodopsin have been studied by ab initio quantum chemical methods. The various factors such as conjugation with a linear system, electron delocalization of the guanidine type, cis-trans isomerism, geometry distortion and hydrogen bonding with charged groups can influence the properties of a given chemical group. Several systems are studied at 4-31G and STO-3G levels. Some of the Schiff-base analogues and guanidine type molecules are characterized by their molecular orbital diagrams, energy levels and the nature of charge distribution. Also, the effects of the above-mentioned factors on proton affinity are studied. It is hoped that the values thus obtained can be helpful in evaluating various structural models for proton transfer.

An ab initio study of (CH⋯X)+ hydrogen bonds including biological systems

Abstract Ionic hydrogen bonds are stronger than neutral hydrogen bonds. Ionic hydrogen bonds, with carbon as the donor, are investigated here at the ab initio level. The hydrogen bond energy for systems with sp3 carbon as donor is 9–15 kcal mol−1 and the C⋯X distance is ∼2.75 A, whereas the hydrogen bonds involving sp2 and sp carbons are much stronger and shorter. Biological systems, such as protonated histidine and acetylcholine have been studied. The CH⋯O hydrogen bond interaction in zwitterionic glycine was found to be substantial. In addition electrostatic potential studies on the donor systems were done to allow qualitative comparison of the hydrogen bond energies.