Enhanced Prediction Accuracy of Protein Secondary Structure Using Hydrogen Exchange Fourier Transform Infrared Spectroscopy

Abstract

A novel equilibrium hydrogen exchange Fourier transform IR (HX-FTIR) spectroscopy method for predicting secondary structure content was employed using spectra obtained for a training set of 23 globular proteins. The IR bandshape and frequency changes resulting from controlled levels of H–D exchange were observed to be protein-dependent. Their analysis revealed these variations to be partly correlated to secondary structure. For each protein, a set of 6 spectra was measured with a systematic variation of the solvent H–D ratio and was subjected to factor analysis. The most significant component spectra for each protein, representing independent aspects of the spectral response to deuteration, were each subjected to a second factor analysis over the entire training set. Restricted multiple regression (RMR) analysis using the loadings of the principal components from 19 of these H–D analyses revealed an improvement in prediction accuracy compared with conventional bandshape-based analyses of FTIR data. Nearly a factor of 2 reduction in error for prediction of helix fractions was found using s1, the average spectral response for the H–D set. In some cases, significant error reduction for prediction of minor components was found using higher factors. Using the same analytical methods, prediction errors with this new deuteration–response–FTIR method were shown to be even better than those obtained by use of electronic circular dichroism (ECD) data for helix predictions and to be significantly lower for ECD-based sheet prediction, making these the best secondary structure predictions obtained with the RMR method. Tests of a limited variable selection scheme showed further improvements, consistent with previous results of this approach using ECD data.