Residue specific contributions to stability and activity inferred from saturation mutagenesis and deep sequencing.
Multiple methods currently exist for rapid construction and screening of single-site saturation mutagenesis (SSM) libraries in which every codon or nucleotide in a DNA fragment is individually randomized. Nucleotide sequences of each library member before and after screening or selection can be obtained through deep sequencing. The relative enrichment of each mutant at each position provides information on its contribution to protein activity or ligand-binding under the conditions of the screen. Such saturation scans have been applied to diverse proteins to delineate hot-spot residues, stability determinants, and for comprehensive fitness estimates. The data have been used to design proteins with enhanced stability, activity and altered specificity relative to wild-type, to test computational predictions of binding affinity, and for protein model discrimination. Future improvements in deep sequencing read lengths and accuracy should allow comprehensive studies of epistatic effects, of combinational variation at multiple sites, and identification of spatially proximate residues.
Molecular Biophysics Unit, Indian Institute of Science, Bangalore 560 012, India.
Arti
A
Tripathi
Molecular Biophysics Unit, Indian Institute of Science, Bangalore 560 012, India; Jawaharlal Nehru Centre for Advanced Scientific Research, Jakkur, Bangalore 560 064, India. Electronic address: varadar@mbu.iisc.ernet.in.
Raghavan
R
Varadarajan
D000818:Animals; D015723:Gene Library; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods*; D006801:Humans; D008958:Models, Molecular; D016296:Mutagenesis; D011506:Proteins / Q000737:chemistry / Q000235:genetics*
D011506:Proteins