Abstract

Despite impressive progress in protein engineering and design, our ability to create new and efficient enzyme activities remains a laborious and time-consuming endeavor. In the past few years, intricate combinations of rational mutagenesis, directed evolution and computational methods have paved the way to exciting engineering examples and are now offering a new perspective on the structural requirements of enzyme activity. However, these structure-function analyses are usually guided by the time-averaged static models offered by enzyme crystal structures, which often fail to describe the functionally relevant ‘invisible states’ adopted by proteins in space and time. To alleviate such limitations, NMR relaxation dispersion experiments coupled to mutagenesis studies have recently been applied to the study of enzyme catalysis, effectively complementing ‘structure-function’ analyses with ‘flexibility-function’ investigation. In addition to offering quantitative, site-specific information to help characterize residue motion, these NMR methods are now being applied to enzyme engineering purposes, providing a powerful tool to help characterize the effects of controlling long-range networks of flexible residues affecting enzyme function. Recent advancements in this emerging field are presented here, with particular attention to mutagenesis reports highlighting the relevance of NMR relaxation dispersion tools in enzyme engineering.

Keywords: CPMG, enzyme catalysis, NMR spectroscopy, protein engineering, relaxation dispersion, residue motion, Protein Motions, NMR Relaxation, rational mutagenesis, structure-function, flexibility-function, dispersion tools, biocatalysts, semi-random mutagenesis, de novo, nanoscale machines, dihydrofolate reductase, adenylate kinase, amino acid networks, enablers, disruptors, dispersion experiments, Cyclophilin A, Pin1, Ribonuclease A, drug development and nanotechnologyCPMG, enzyme catalysis, NMR spectroscopy, protein engineering, relaxation dispersion, residue motion, Protein Motions, NMR Relaxation, rational mutagenesis, structure-function, flexibility-function, dispersion tools, biocatalysts, semi-random mutagenesis, de novo, nanoscale machines, dihydrofolate reductase, adenylate kinase, amino acid networks, enablers, disruptors, dispersion experiments, Cyclophilin A, Pin1, Ribonuclease A, drug development and nanotechnology