Dietrich, Susanne and Borst, Nadine and Schlee, Sandra and Schneider, Daniel and Janda, Jan-Oliver and Sterner, Reinhard and Merkl, Rainer (2012) Experimental Assessment of the Importance of Amino Acid Positions Identified by an Entropy-Based Correlation Analysis of Multiple-Sequence Alignments. BIOCHEMISTRY, 51 (28). pp. 5633-5641. ISSN 0006-2960,
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The analysis of a multiple-sequence alignment (MSA) with correlation methods identifies pairs of residue positions whose occupation with amino acids changes in a concerted manner. It is plausible to assume that positions that are part 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 large number of significant correlations in which k is involved. 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 nonconserved residues with alanine or the prevalent residue of the MSA. As a control, five residues with conn(k) values of zero were chosen randomly and replaced with 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 (Delta T-M(app) = 14 degrees 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 degrees C. For the sIGPS control proteins, the observed activity and stability decreases 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.
| Item Type: | Article |
|---|---|
| Uncontrolled Keywords: | INDOLE-3-GLYCEROL PHOSPHATE SYNTHASE; FUNCTIONALLY IMPORTANT RESIDUES; SITE-DIRECTED MUTAGENESIS; SULFOLOBUS-SOLFATARICUS; ANTHRANILATE PHOSPHORIBOSYLTRANSFERASE; (BETA-ALPHA)(8)-BARREL ENZYME; MUTATIONAL ANALYSIS; CATALYTIC-ACTIVITY; PROTEIN STABILITY; HIGH-THROUGHPUT; |
| Subjects: | 500 Science > 570 Life sciences |
| Divisions: | Biology, Preclinical Medicine > Institut für Biophysik und physikalische Biochemie > Prof. Dr. Reinhard Sterner Biology, Preclinical Medicine > Institut für Biophysik und physikalische Biochemie > Prof. Dr. Rainer Merkl |
| Depositing User: | Dr. Gernot Deinzer |
| Date Deposited: | 11 May 2020 05:10 |
| Last Modified: | 11 May 2020 05:10 |
| URI: | https://pred.uni-regensburg.de/id/eprint/18437 |
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