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/β-mixed or β-structure-rich proteins. The problem arises from the spectral diversity of β-structures, which has hitherto been considered as an intrinsic limitation of the technique. The predictions are less reliable for proteins of unusual β-structures such as membrane proteins, protein aggregates, and amyloid fibrils. Here, we show that the parallel/antiparallel orientation and the twisting of the β-sheets account for the observed spectral diversity. We have developed a method called β-structure selection (BeStSel) for the secondary structure estimation that takes into account the twist of β-structures. This method can reliably distinguish parallel and antiparallel β-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 | secondary structure determination | protein fold | protein aggregation | amyloid O ptically active macromolecules, such as proteins, exhibit differential absorption of circular polarized light. The far-UV circular dichroism (CD) spectroscopy of proteins and peptides (180-250 nm) is predominantly based on the excitation of electronic transitions in amide groups. The peptide backbone forms characteristic secondary structures such as α-helices, β-pleated sheets, turns, and disordered sections with specific Φ, Ψ dihedral angles and H-bond patterns affecting the CD spectrum (1). CD has been exploited for protein folding and stability assays, intermolecular interactions, and ligand binding studies, and has recently been applied in the investigations of protein disorder (2, 3). Synchrotron radiation CD (SRCD) spectroscopy is an emerging technique complementary to small-angle X-ray scattering or infrared spectroscopy, synergistic to biochemical and biophysical assays characterizing the protein folding state. SRCD extends the limits of conventional CD spectroscopy by broadening the spectral range, increasing the signal-to-noise ratio, and accelerating the data acquisition, in the presence of absorbing components (buffers, salts, etc.) (4). Additionally, SRCD has the capability of time-resolved and stopped-flow measurements as well as hig...

Circular dichroism (CD) spectroscopy is a widely used method to study the protein secondary structure. However, for decades, the general opinion was that the correct estimation of β-sheet content is challenging because of the large spectral and structural diversity of β-sheets. Recently, we showed that the orientation and twisting of β-sheets account for the observed spectral diversity, and developed a new method to estimate accurately the secondary structure (PNAS, 112, E3095). BeStSel web server provides the Beta Structure Selection method to analyze the CD spectra recorded by conventional or synchrotron radiation CD equipment. Both normalized and measured data can be uploaded to the server either as a single spectrum or series of spectra. The originality of BeStSel is that it carries out a detailed secondary structure analysis providing information on eight secondary structure components including parallel-β structure and antiparallel β-sheets with three different groups of twist. Based on these, it predicts the protein fold down to the topology/homology level of the CATH protein fold classification. The server also provides a module to analyze the structures deposited in the PDB for BeStSel secondary structure contents in relation to Dictionary of Secondary Structure of Proteins data. The BeStSel server is freely accessible at http://bestsel.elte.hu.

Circular Dichroism (CD) spectroscopy is a long-established technique for studying protein secondary structures in solution. Empirical analyses of CD data rely on the availability of reference datasets comprised of far-UV CD spectra of proteins whose crystal structures have been determined. This article reports on the creation of a new reference dataset which effectively covers both secondary structure and fold space, and uses the higher information content available in synchrotron radiation circular dichroism (SRCD) spectra to more accurately predict secondary structure than has been possible with existing reference datasets. It also examines the effects of wavelength range, structural redundancy and different means of categorizing secondary structures on the accuracy of the analyses. In addition, it describes a novel use of hierarchical cluster analyses to identify protein relatedness based on spectral properties alone. The databases are shown to be applicable in both conventional CD and SRCD spectroscopic analyses of proteins. Hence, by combining new bioinformatics and biophysical methods, a database has been produced that should have wide applicability as a tool for structural molecular biology.

Amphipols (APols) are short amphipathic polymers that can substitute for detergents to keep integral membrane proteins (MPs) water soluble. In this review, we discuss their structure and solution behavior; the way they associate with MPs; and the structure, dynamics, and solution properties of the resulting complexes. All MPs tested to date form water-soluble complexes with APols, and their biochemical stability is in general greatly improved compared with MPs in detergent solutions. The functionality and ligand-binding properties of APol-trapped MPs are reviewed, and the mechanisms by which APols stabilize MPs are discussed. Applications of APols include MP folding and cell-free synthesis, structural studies by NMR, electron microscopy and X-ray diffraction, APol-mediated immobilization of MPs onto solid supports, proteomics, delivery of MPs to preexisting membranes, and vaccine formulation.

CDtool is a software package written to facilitate circular dichroism (CD) spectroscopic studies on both conventional lab-based instruments and synchrotron beamlines. It takes format-independent input data from any type of CD instrument, enables a wide range of standard and advanced processing methods, and, in a single user-friendly graphics-based package, takes raw data through the entire processing procedure and, importantly, uses data-mining techniques to retain in the final output all the information associated with the processing. It permits the facile comparison of data obtained from different instruments without the need for reformatting and displays it in graphical formats suitable for publication. It also includes the ability to automatically archive the processed data. This latter feature may be especially useful in light of recent funding institution directives with regard to data sharing and archiving and requirements for ''good practice'' and ''traceability'' within the pharmaceutical industry. In addition, CDtool includes a means of interfacing with protein data bank coordinate files and calculating secondary structures from them using alternate definitions and algorithms. This feature, along with a function that permits the facile production of new reference databases, enables the creation of specialized databases for secondary structural analyses of specific types of proteins. Thus the CDtool software not only enables rapid data processing and analyses but also includes many enhanced features not available in other CD data processing/analysis packages. Ó 2004 Elsevier Inc. All rights reserved.Keywords: Circular dichroism spectroscopy; Synchrotron radiation circular dichroism (SRCD); Archive; Data processing; Software; PDB Rapid data collection is one of the advantages of using circular dichroism (CD) spectroscopy for the analysis of protein structures. It is the subsequent data processing and analysis steps that can be surprisingly time consuming. This discrepancy becomes even more acute for synchrotron radiation-based circular dichroism (SRCD) 1 spectra, which can be accumulated on a very short timescale.Currently several different software packages are required for processing, comparing, and analyzing CD data. Data collection and some processing software are usually supplied with commercial CD instruments and each SRCD beamline has developed its own data collection software. The data processing functions from the various instruments tend to differ considerably, which makes cross-instrument processing of data, and thus comparisons, extremely difficult. To surmount these problems in the past, a very simple generic program (SUPER3) was developed to include a range of processing functions [1] and simple analyses, but this software does not now adequately meet the needs of ever more sophisticated data collection procedures.Analysis tools developed for CD spectra include a wide range of secondary structure calculation algorithms [2-6] which often require reformatting, rescaling, and other man...

Antiherpes therapies are principally targeted at viral thymidine kinases and utilize nucleoside analogs, the triphosphates of which are inhibitors of viral DNA polymerase or result in toxic effects when incorporated into DNA. The most frequently used drug, aciclovir (Zovirax), is a relatively poor substrate for thymidine kinase and high-resolution structural information on drugs and other molecules binding to the target is therefore important for the design of novel and more potent chemotherapy, both in antiherpes treatment and in gene therapy systems where thymidine kinase is expressed. Here, we report for the first time the binary complexes of HSV-1 thymidine kinase (TK) with the drug molecules aciclovir and penciclovir, determined by X-ray crystallography at 2.37 A resolution. Moreover, from new data at 2.14 A resolution, the refined structure of the complex of TK with its substrate deoxythymidine (R = 0.209 for 96% of all data) now reveals much detail concerning substrate and solvent interactions with the enzyme. Structures of the complexes of TK with four halogen-containing substrate analogs have also been solved, to resolutions better than 2.4 A. The various TK inhibitors broadly fall into three groups which together probe the space of the enzyme active site in a manner that no one molecule does alone, so giving a composite picture of active site interactions that can be exploited in the design of novel compounds.

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Synchrotron radiation circular dichroism (SRCD) is an emerging technique in structural biology with particular value in protein secondary structure analyses since it permits the collection of data down to much lower wavelengths than conventional circular dichroism (cCD) instruments. Reference database spectra collected on different SRCD instruments in the future as well as current reference datasets derived from cCD spectra must be compatible. Therefore there is a need for standardization of calibration methods to ensure quality control. In this study, magnitude and optical rotation measurements on four cCD and three SRCD instruments were compared at 192.5, 219, 290 and 490 nm. At high wavelengths, all gave comparable results, however, at the lower wavelengths, some variations were observable. The consequences of these differences on the spectrum, and the calculated secondary structure, of a representative protein (myoglobin) are demonstrated. A method is proposed for standardising spectra obtained on any CD instrument, conventional or synchrotron‒based, with respect to existing and future databases.