MOTIVATION: Circular dichroism (CD) spectroscopy is extensively utilized for determining the percentages of secondary structure content present in proteins. However, although a large contributor, secondary structure is not the only factor that influences the shape and magnitude of the CD spectrum produced. Other structural features can make contributions so an entire protein structural conformation can give rise to a CD spectrum. There is a need for an application capable of generating protein CD spectra from atomic coordinates. However, no empirically derived method to do this currently exists. RESULTS: PDB2CD has been created as an empirical-based approach to the generation of protein CD spectra from atomic coordinates. The method utilizes a combination of structural features within the conformation of a protein; not only its percentage secondary structure content, but also the juxtaposition of these structural components relative to one another, and the overall structure similarity of the query protein to proteins in our dataset, the SP175 dataset, the 'gold standard' set obtained from the Protein Circular Dichroism Data Bank (PCDDB). A significant number of the CD spectra associated with the 71 proteins in this dataset have been produced with excellent accuracy using a leave-one-out cross-validation process. The method also creates spectra in good agreement with those of a test set of 14 proteins from the PCDDB. The PDB2CD package provides a web-based, user friendly approach to enable researchers to produce CD spectra from protein atomic coordinates. AVAILABILITY AND IMPLEMENTATION: http://pdb2cd.cryst.bbk.ac.uk CONTACT: [email protected] information: Supplementary data are available at Bioinformatics online.

SUMMARY: The defined secondary structure of proteins method is often considered the gold standard for assignment of secondary structure from three-dimensional coordinates. However, there are alternative methods. '2Struc: The Secondary Structure Server' has been created as a single point of access for eight different secondary structure assignment methods. It has been designed to enable comparisons between methods for analyzing the secondary structure content for a single protein. It also includes a second functionality, 'Compare-the-Protein' to enable comparisons of the secondary structure features from any one method to be made within a collection of nuclear magnetic resonance models, or between the crystal structures of two different proteins. AVAILABILITY: http://2struc.cryst.bbk.ac.uk CONTACT: [email protected] SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

BACKGROUND: A number of sequence-based methods exist for protein secondary structure prediction. Protein secondary structures can also be determined experimentally from circular dichroism, and infrared spectroscopic data using empirical analysis methods. It has been proposed that comparable accuracy can be obtained from sequence-based predictions as from these biophysical measurements. Here we have examined the secondary structure determination accuracies of sequence prediction methods with the empirically determined values from the spectroscopic data on datasets of proteins for which both crystal structures and spectroscopic data are available. RESULTS: In this study we show that the sequence prediction methods have accuracies nearly comparable to those of spectroscopic methods. However, we also demonstrate that combining the spectroscopic and sequences techniques produces significant overall improvements in secondary structure determinations. In addition, combining the extra information content available from synchrotron radiation circular dichroism data with sequence methods also shows improvements. CONCLUSION: Combining sequence prediction with experimentally determined spectroscopic methods for protein secondary structure content significantly enhances the accuracy of the overall results obtained.

MOTIVATION: Hydrogen bonds are one of the most important inter-atomic interactions in biology. Previous experimental, theoretical and bioinformatics analyses have shown that the hydrogen bonding potential of amino acids is generally satisfied and that buried unsatisfied hydrogen-bond-capable residues are destabilizing. When studying mutant proteins, or introducing mutations to residues involved in hydrogen bonding, one needs to know whether a hydrogen bond can be maintained. Our aim, therefore, was to develop a rapid method to evaluate whether a sidechain can form a hydrogen-bond. RESULTS: A novel knowledge-based approach was developed in which the conformations accessible to the residues involved are taken into account. Residues involved in hydrogen bonds in a set of high resolution crystal structures were analyzed and this analysis is then applied to a given protein. The program was applied to assess mutations in the tumour-suppressor protein, p53. This raised the number of distinct mutations identified as disrupting sidechain-sidechain hydrogen bonding from 181 in our previous analysis to 202 in this analysis.

BACKGROUND: Circular Dichroism (CD) spectroscopy is a widely used method for studying protein structures in solution. Modern synchrotron radiation CD (SRCD) instruments have considerably higher photon fluxes than do conventional lab-based CD instruments, and hence have the ability to routinely measure CD data to much lower wavelengths. Recently a new reference dataset of SRCD spectra of proteins of known structure, designed to cover secondary structure and fold space, has been produced which includes low wavelength (vacuum ultraviolet - VUV) data. However, the existing algorithms used to calculate protein secondary structures from CD data have not been designed to take optimal advantage of the additional information in these low wavelength data. RESULTS: In this study, we have optimised secondary structure calculation methods based on the low wavelength CD data by examining existing algorithms and secondary structure assignment schemes, and then developing new methods which have produced clear improvements in prediction accuracy, especially for beta-sheet components. We have further shown that if precise measurements of protein concentrations, and therefore spectral magnitudes, are not available, the inclusion of the low wavelength data will significantly improve the analyses. However, we have also demonstrated that the new reference dataset, methods, and assignments can also improve the analyses of conventional circular dichroism data, even if the low wavelength data is not available. CONCLUSION: VUV CD data include important information on protein structure which can be exploited with the algorithms and methodologies described.

MOTIVATION: Circular dichroism (CD) spectroscopy has become established as a key method for determining the secondary structure contents of proteins which has had a significant impact on molecular biology. Many excellent mathematical protocols have been developed for this purpose and their quality is above question. However, reference database sets of proteins, with CD spectra matched to secondary structure components derived from X-ray structures, provide the key resource for this task. These databases were created many years ago, before most CD spectrophotometers became standardized and before it was commonplace to validate X-ray structures prior to publication. The analyses presented here were undertaken to investigate the overall quality of these reference databases in light of their extensive usage in determining protein secondary structure content from CD spectra. RESULTS: The analyses show that there are a number of significant problems associated with the CD reference database sets in current use. There are disparities between CD spectra for the same protein collected by different groups. These include differences in magnitudes, peak positions or both. However, many current reference sets are now amalgamations of spectra from these groups, introducing inconsistencies that can lead to inaccuracies in the determination of secondary structure components from the CD spectra. A number of the X-ray structures used fall short on the validation criteria now employed as standard for structure determination. Many have substantial percentages of residues in the disallowed regions of the Ramachandran plot. Hence their calculated secondary structure components, used as a foundation for the reference databases, are likely to be in error. Additionally, the coverage of secondary structure space in the reference datasets is poorly correlated to the secondary structure components found in the Protein Data Bank. A conclusion is that a new reference CD database with cross-correlated, machine-independent CD spectra and validated X-ray structures that cover more secondary structure components, including diverse protein folds, is now needed. However, that reasonably accurate values for the secondary structure content of proteins can be determined from spectra is a testament to CD spectroscopy being a very powerful technique.