Experimental assessment of the importance of amino acid positions identified by an entropy-based correlation analysis of multiple sequence alignments

Abstract

The analysis of a multiple sequence alignment (MSA) with correlation methods identifies for a protein pairs of residue-positions whose occupation with amino acids change in a concerted manner. It is plausible to assume that positions that are a member of many such correlation pairs are important for protein function or stability. We have used the algorithm H2r to identify positions k in the MSAs of the enzymes anthranilate phosphoribosyl transferase (AnPRT) and indole-3-glycerol phosphate synthase (IGPS) that show a high conn(k) value, i.e. a high number of significant correlations k is involved in. The importance of the identified residues was experimentally validated by performing mutagenesis studies with sAnPRT and sIGPS from the archaeon Sulfolobus solfataricus. For sAnPRT, five H2r-mutant proteins were generated by replacing non-conserved residues with alanine or the prevalent residue of the MSA. As control, five residues with conn(k) values of zero were chosen randomly and replaced by alanine. The catalytic activities and conformational stabilities of the H2r and control mutant proteins were analyzed by steady-state enzyme kinetics and thermal unfolding studies. Compared to wild-type sAnPRT, the catalytic efficiencies (k(cat)/K(M)) were largely unaltered. In contrast, the apparent thermal unfolding temperature (T(M)(app)) was lowered in most proteins. Remarkably, the strongest observed destabilization (ΔT(M)(app) = 14 °C) was caused by the V284A exchange, which pertains to the position with the highest correlation signal (conn(k) = 11). For sIGPS, six H2r mutant and four control proteins with alanine exchanges were generated and characterized. The k(cat)/K(M) values of four H2r-mutant proteins were reduced between 13- and 120-fold, and their T(M)(app) values were decreased by up to 5 °C. For the sIGPS control proteins, the observed activity and stability drops were much less severe. Our findings demonstrate that positions with high conn(k) values have an increased probability of being important for enzyme function or stability.