Infrared absorbances of protein side chains.

The spectral parameters of amino acid residue side chain and peptide bond absorptions in the region 1800-1440 cm-1 have been obtained by using an inverse matrix method applied to the infrared spectra of 42 amino acids, dipeptides, and higher peptides in aqueous solution. In addition the pH-dependent extinction coefficients of the amino acid and peptide COO-/NH3+ end groups were derived. It is shown that the secondary structure prediction accuracy of proteins by multivariate data analysis methods increases slightly, if the side chain absorbances of the residues asparagine, glutamine, aspartic acid, glutamic acid, arginine, tyrosine, and lysine are subtracted from the amide I and amide II region.

Prediction of secondary structures of proteins using the sequence and spectroscopical data.

An algorithm is presented which modifies the parameter of given methods of prediction of secondary protein structures by comparing the predictions with the frequency of secondary structures derived from infrared spectra in a way that the predictions align to the given data. Depending on the prediction method and accuracy of the given secondary structures a 1-6% increase in accuracy can be reached. The algorithm compares the difference between the predicted and real frequency of a given secondary structure in the protein and modifies accordingly the parameter used in the prediction method in order to give a new, more accurate prediction. A correlation between the accuracy of the prediction and increasing correctness between the prediction and infrared data was found using a set of 39 proteins.

Electronic influences on an infrared detector signal: nonlinearity and amplification.

For mercury-cadmium-telluride detectors, frequently used in Fourier transform IR spectroscopy, the recorded signal is a nonlinear function of the light intensity. This behavior depends on a series resistor in the electronic circuit and thus the illumination of the detector. This nonlinearity must be accounted for to avoid spectroscopic errors. The results of theoretically calculating the effect permit a correction that can be applied, with corresponding lower accuracy, even after a phase correction. Also the use of the amplification stages does influence the phase of the signal electronically. For an accurate nonlinearity correction, compensation of the amplification of the analog signal is advisable.

Phase correction in Fourier transform spectroscopy: subsequent displacement correction and error limit.

In Fourier transform spectroscopy interferograms are apodized prior to the transformation and the calculated spectrum is phase corrected. If the zero point for the Fourier transformation is displaced, a phase error occurs because the apodization remains fixed while the interferogram is shifted by the phase correction. A theoretical description of this effect is derived and an algorithm for subsequent removal is provided. It is also reported that fixing the zero point to solely the sampling positions leads to error in the band shape that by far exceeds the noise and therefore limits the spectroscopic accuracy.

Secondary structure determination of proteins in aqueous solution by infrared spectroscopy: a comparison of multivariate data analysis methods.

The accuracy of the secondary structure prediction from an infrared spectra data base of 39 proteins with known X-ray structure was investigated by different methods of multivariate data analysis. The best agreements with the secondary structure determined by X-ray crystallography are obtained if both the amide I and amide II bands are used for calibration. With optimized parameters the methods singular value decomposition, partial least squares, and ridge regression yield similar results. As judged by the standard error of prediction, the secondary structure elements helix and beta-sheet can be predicted with the highest accuracy. Small data sets of less than 20 protein spectra, which exhibit the variance in secondary structure content of the whole set, can pretend an increased prediction accuracy only if column cross-validation is used as reference; however, with these calibration sets the average secondary structure prediction of all 39 proteins is debased. The hydrogen-bonded turns or bridges are predicted with higher accuracy than the assigned secondary structure types helix and beta-sheet.

A correlation between thermal stability and structural features of staphylokinase and selected mutants: a Fourier-transform infrared study.

Variants of recombinant staphylokinase (Sak) were investigated by Fourier-transform infrared spectroscopy: Sak (wild type), Sak-M26A, Sak-M26L, and Sak-G34S/R36G/R43H (Sak-B). Estimation of the secondary structure and hydrogen-deuterium exchange experiments revealed the existence of fast-exchanging and strongly solvent-exposed fractions of the helical structures in the two samples Sak and Sak-M26L. These two samples are also thermally less stable with unfolding transition temperatures of 43.7 degrees C (Sak) and 43.5 degrees C (Sak-M26L), respectively. On contrast, Sak-M26A and Sak-G34S/R36G/R43H have a slower hydrogen-deuterium exchange, have a smaller solvent-exposed portion of the helical part, and are more resistant against thermal unfolding; the transition temperatures are 51.7 degrees C and 59.3 degrees C, respectively. The secondary structure analysis was performed by two different approaches, by curve-fitting after band narrowing and by pattern recognition (factor analysis) based upon reference spectra of proteins with known crystal structure. Within the limits of the used methods, we are unable to detect significant differences in the secondary structure of the four variants of Sak. According to the results of the factor analysis, the portions of secondary structure elements were obtained to 16-20% alpha-helix, 28-30% beta-sheet, 23-27% turns, 28-30% irregular (random) and other structure. The sharp differences in the specific plasminogen-activating capacity (Sak, Sak-G34S/R36G/R43H and Sak-M26L are fully active, but Sak-M26A does not form a stable complex with plasminogen) are not reflected in the structural features revealed by the infrared spectra of this study.