Accurate secondary structure prediction and fold recognition for circular dichroism spectroscopy.

Circular dichroism (CD) spectroscopy is a widely used technique for the study of protein structure. Numerous algorithms have been developed for the estimation of the secondary structure composition from the CD spectra. These methods often fail to provide acceptable results on α/ß-mixed or ß-structure-rich proteins. The problem arises from the spectral diversity of ß-structures, which has hitherto been considered as an intrinsic limitation of the technique. The predictions are less reliable for proteins of unusual ß-structures such as membrane proteins, protein aggregates, and amyloid fibrils. Here, we show that the parallel/antiparallel orientation and the twisting of the ß-sheets account for the observed spectral diversity. We have developed a method called ß-structure selection (BeStSel) for the secondary structure estimation that takes into account the twist of ß-structures. This method can reliably distinguish parallel and antiparallel ß-sheets and accurately estimates the secondary structure for a broad range of proteins. Moreover, the secondary structure components applied by the method are characteristic to the protein fold, and thus the fold can be predicted to the level of topology in the CATH classification from a single CD spectrum. By constructing a web server, we offer a general tool for a quick and reliable structure analysis using conventional CD or synchrotron radiation CD (SRCD) spectroscopy for the protein science research community. The method is especially useful when X-ray or NMR techniques fail. Using BeStSel on data collected by SRCD spectroscopy, we investigated the structure of amyloid fibrils of various disease-related proteins and peptides.

Circular dichroism in drug discovery and development: an abridged review.

Chirality plays a fundamental role in determining the pharmacodynamic and pharmacokinetic properties of drugs, and contributes significantly to our understanding of the mechanisms that lie behind biorecognition phenomena. Circular dichroism spectroscopy is the technique of choice for determining the stereochemistry of chiral drugs and proteins, and for monitoring and characterizing molecular recognition phenomena in solution. The role of chirality in our understanding of recognition phenomena at the molecular level is discussed here via several selected systems of interest in the drug discovery and development area. The examples were selected in order to underline the utility of circular dichroism in emerging studies of protein-protein interactions in biological context. In particular, the following aspects are discussed here: the relationship between stereochemistry and pharmacological activity--stereochemical characterization of new leads and drugs; stereoselective binding of leads and drugs to target proteins--the binding of drugs to serum albumins; conformational transitions of peptides and proteins of physiological relevance, and the stereochemical characterization of therapeutic peptides.

Structural and functional alterations of two multidomain oxidoreductases induced by guanidine hydrochloride.

The unfolding and refolding of two multidomain oxidoreductases, bovine liver catalase and flavoprotein bovine milk xanthine oxidase (XO), have been analyzed by fluorescence spectroscopy, circular dichroism, and activity measurements. Two intermediates, a partially folded active dimer disassembled from the native tetramer and a partially folded inactivated monomer, are found to exist in the conformational changes of catalase induced by guanidine hydrochloride (GdnHCl). Similarly, two intermediates, an active, compacted intermediate bound by flavin adenine dinucleotide (FAD) partially and an inactive flexible intermediate with FAD completely dissociated, exist in the conformational changes of XO induced by GdnHCl. The activity regains completely and an enhancement in activity compared with the native catalase or native XO is observed by dilution of catalase or XO incubated with GdnHCl at concentrations not >0.5 or 1.8 M into the refolding buffer, but the yield of reactivation for catalase or XO is zero when the concentration of GdnHCl is >1.5 or 3.0 M. The addition of FAD provides a remarkable protection against the inactivation of XO by GdnHCl under mild denaturing conditions, and the conformational change of XO is irreversible after FAD has been removed in the presence of a strong denaturing agent. These findings provide impetus for exploring the influences of cofactors such as FAD on the structure-function relationship of xanthine oxidoreductases.

Vibrational circular dichroism spectroscopy of solid polymer films: effects of sample orientation.

Vibrational circular dichroism (VCD) spectra of anisotropic thin solid samples are often superimposed with large contributions of linear birefringence and linear dichroism. In this study a theoretical approach is given on how to extract the true VCD spectrum out of such superimposed spectra. To verify this approach, the VCD spectra of achiral polymer films were examined. The polymers are supposed to give a zero line as VCD spectrum after eliminating the linear contributions. Applying our approach, in which four VCD spectra in different but selected sample orientations are recorded, and calculating their average, leads to the expected result, i.e., a zero line for achiral polymers. The advantage of this method for the elimination of artifacts from solid-state VCD spectra is that no further measurements are required (e.g., linear dichroism measurements or the determination of the orientation with the maximum anisotropy).

K2D2: estimation of protein secondary structure from circular dichroism spectra.

BACKGROUND: Circular dichroism spectroscopy is a widely used technique to analyze the secondary structure of proteins in solution. Predictive methods use the circular dichroism spectra from proteins of known tertiary structure to assess the secondary structure contents of a protein with unknown structure given its circular dichroism spectrum. RESULTS: We developed K2D2, a method with an associated web server to estimate protein secondary structure from circular dichroism spectra. The method uses a self-organized map of spectra from proteins with known structure to deduce a map of protein secondary structure that is used to do the predictions. CONCLUSION: The K2D2 server is publicly accessible at http://www.ogic.ca/projects/k2d2/. It accepts as input a circular dichroism spectrum and outputs the estimated secondary structure content (alpha-helix and beta-strand) of the corresponding protein, as well as an estimated measure of error.

Multivariate curve resolution applied to the simultaneous analysis of electrochemical and spectroscopic data: study of the Cd(II)/glutathione-fragment system by voltammetry and circular dichroism spectroscopy.

Multivariate curve resolution with alternating least squares (MCR-ALS) is applied for the first time to the simultaneous analysis of electrochemical and spectroscopic data. Then, a data analysis is done with augmented matrices constituted by Differential Pulse Polarography and Circular Dichroism data submatrices. The use of proper, and different for each submatrix, constrains in the iterative ALS optimization allows to obtain chemically meaningful results constituted by a common matrix containing the concentration profiles, and two matrices with the pure electrochemical and spectroscopic signals. MCR-ALS is applied to the study of the complexation of Cd by Cys-Gly, a glutathione-fragment of great interest for understanding metal-phytochelatins complexation.

Circular dichroism spectroscopy for the study of protein-ligand interactions.

Circular dichroism (CD) is the difference in absorption of left and right circularly polarized light, usually by a solution containing the molecules of interest. A signal is only measured for chiral molecules such as proteins. A CD spectrum provides information about the bonds and structures responsible for this chirality. When a small molecule (or ligand) binds to a protein, it acquires an induced CD (ICD) spectrum through chiral perturbation to its structure or electron rearrangements. The wavelengths of this ICD are determined by the ligand's own absorption spectrum, and the intensity of the ICD spectrum is determined by the strength and geometry of its interaction with the protein. Thus, ICD can be used to probe the binding of ligands to proteins. This chapter outlines protein CD and ICD, together with some of the issues relating to experimental design and implementation.

A linear correlation between the energetics of allosteric communication and protein flexibility in the Escherichia coli cyclic AMP receptor protein revealed by mutation-induced changes in compressibility and amide hydrogen-deuterium exchange.

Amino acid substitutions at distant sites in the Escherichia coli cyclic AMP receptor protein (CRP) have been shown to affect both the nature and magnitude of the energetics of cooperativity of cAMP binding, ranging from negative to positive. In addition, the binding to DNA is concomitantly affected. To correlate the effects of amino acid substitutions on the functional energetics and global structural properties in CRP, the partial specific volume (v(o)), the coefficient of adiabatic compressibility (beta(s)(o)), and the rate of amide proton exchange were determined for the wild-type and eight mutant CRPs (K52N, D53H, S62F, T127L, G141Q, L148R, H159L, and K52N/H159L) by using sound velocity, density measurements, and hydrogen-deuterium exchange as monitored by Fourier transform infrared spectroscopy at 25 degrees C. These mutations induced large changes in v(o) (0.747-0.756 mL/g) and beta(s)(o) (6.89-9.68 Mbar(-1)) compared to the corresponding values for wild-type CRP (v(o)= 0.750 mL/g and beta(s)(o)= 7.98 Mbar(-1)). These changes in global structural properties correlated with the rate of amide proton exchange. A linear correlation was established between beta(s)(o) and the energetics of cooperativity of binding of cAMP to the high-affinity sites, regardless of the nature of cooperativity, be it negative or positive. This linear correlation indicates that the nature and magnitude of cooperativity are a continuum. A similar linear correlation was established between compressibility and DNA binding affinity. In addition, linear correlations were also found among the dynamics of CRP and functional energetics. Double mutation (K52N/H159L) at positions 52 and 159, whose alpha-carbons are separated by 34.6 A, showed nonadditive effects on v(o) and beta(s)(o). These results demonstrate that a small alteration in the local structure due to amino acid substitution is dramatically magnified in the overall protein dynamics which plays an important role in modulating the allosteric behavior of CRP.

Calculations of protein circular dichroism from first principles.

Understanding the relationship between the amino acid sequence of a protein and its unique, compact 3D structure is one of the grand challenges in molecular biophysics. One particularly exciting approach is time-resolved electronic circular dichroism (CD) spectroscopy, which offers resolution on a nanosecond (or faster) time scale, although it does not provide the spatial resolution of techniques like X-ray crystallography or NMR. The thrust of our work is to underpin fast time scale spectroscopic studies of protein folding with a stronger theoretical foundation. Ultimately, we seek to use molecular dynamics simulations to study the influence of conformational dynamics and conformational transitions on the electronic CD spectra of proteins. We discuss how improved quantum chemical models of individual chromophores, including aromatic sidechains, can be incorporated into calculations of the electronic structure of proteins and their CD.

Urea-induced sequential unfolding of fibronectin: a fluorescence spectroscopy and circular dichroism study.

Fibronectin (FN) is an extracellular matrix (ECM) protein found soluble in corporal fluids or as an insoluble fibrillar component incorporated in the ECM. This phenomenon implicates structural changes that expose FN binding sites and activate the protein to promote intermolecular interactions with other FN. We have investigated, using fluorescence and circular dichroism spectroscopy, the unfolding process of human fibronectin induced by urea in different ionic strength conditions. At any ionic strength, the equilibrium unfolding data are well described by a four-state equilibrium model N <= => I(1) <= =>I(2) <= => U. Fitting this model to experimental values, we have determined the free energy change for the different steps. We found that the N <= => I(1) transition corresponds to a free energy of 10.5 +/- 0.4 kcal/mol. Comparable values of free energy change are generally associated with a partial unfolding of the type III domain. For the I(1) <= => I(2) transition, the free energy change is 7.6 +/- 0.4 kcal/mol at low ionic strength but is twice as low at high ionic strength. This result is consistent with observations indicating that the complete unfolding of the type III domain from partially unfolded forms necessitates about 5 kcal/mol. The third step, I(2) <= => U, which leads to the complete unfolding of fibronectin, corresponds to a free energy change of 14.4 +/- 0.9 kcal/mol at low ionic strength whereas this energy is again twice as low under high ionic strength conditions. This hierarchical unfolding of fibronectin, as well as the stability of the different intermediates controlled by ionic strength demonstrated here, could be important for the understanding of activation of the matrix assembly.

Absolute configuration determination of chiral molecules in the solution state using vibrational circular dichroism.

Advances in the measurement, calculation, and application of vibrational circular dichroism (VCD) for the determination of absolute configuration are described. The purpose of the review is to provide an up-to-date perspective on the capability of VCD to solve problems of absolute stereochemistry for chiral molecules primarily in the solution state. The scope of the article covers the experimental methods needed for the accurate measurement of VCD spectra and the theoretical steps required to systematically deduce absolute configuration. Determination of absolute configuration of a molecule by VCD requires knowledge of its conformation or conformational distribution, and hence VCD analysis necessarily provides solution-state conformation information, in many cases available by no other method, as an additional benefit. Comparisons of the advantages and limitations of VCD relative to other available chiroptical methods of analysis are also presented.