Reengineering Rate-Limiting, Millisecond Enzyme Motions by Introduction of an Unnatural Amino Acid

• Published in: Biophysical journal Vol. 101; no. 2; pp. 411 - 420
• Main Authors: Watt, Eric D., Rivalta, Ivan, Whittier, Sean K., Batista, Victor S., Loria, J. Patrick
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 20-07-2011; Biophysical Society; The Biophysical Society
• Subjects: Amino acids; Amino Acids - metabolism; Biocatalysis; catalytic activity; Correlation analysis; energy; histidine; hydrogen bonding; Hydrogen bonds; Imidazoles - metabolism; Kinetics; landscapes; Ligands; molecular dynamics; Molecular Dynamics Simulation; Motion; Mutant Proteins - chemistry; nuclear magnetic resonance spectroscopy; Protein; Protein Engineering; Proteins; ribonucleases; Ribonucleases - metabolism; Time Factors
• ISSN: 0006-3495; 1542-0086
• DOI: 10.1016/j.bpj.2011.05.039

Rate-limiting millisecond motions in wild-type (WT) Ribonuclease A (RNase A) are modulated by histidine 48. Here, we incorporate an unnatural amino acid, thia-methylimidazole, at this site (H48C-4MI) to investigate the effects of a single residue on protein motions over multiple timescales and on enzyme catalytic turnover. Molecular dynamics simulations reveal that H48C-4MI retains some crucial WT-like hydrogen bonding interactions but the extent of protein-wide correlated motions in the nanosecond regime is decreased relative to WT. NMR Carr-Purcell-Meiboom-Gill relaxation dispersion experiments demonstrate that millisecond conformational motions in H48C-4MI are present over a similar pH range compared to WT. Furthermore, incorporation of this nonnatural amino acid allows retention of WT-like catalytic activity over the full pH range. These studies demonstrate that the complexity of the protein energy landscape during the catalytic cycle can be maintained using unnatural amino acids, which may prove useful in enzyme design efforts.