Residue specific contributions to stability and activity inferred from saturation mutagenesis and deep sequencing

• Published in: Current opinion in structural biology Vol. 24; pp. 63 - 71
• Main Authors: Tripathi, Arti, Varadarajan, Raghavan
• Format: Journal Article
• Language: English
• Published: England Elsevier Ltd 01.02.2014
• Subjects: Animals; Gene Library; High-Throughput Nucleotide Sequencing - methods; Humans; Models, Molecular; Mutagenesis; Proteins - chemistry; Proteins - genetics
• ISSN: 0959-440X; 1879-033X
• DOI: 10.1016/j.sbi.2013.12.001

•Individual mutant populations in a saturation library derived via deep sequencing.•Enrichment of each single-site mutant, following a selection is measured.•This provides residue specific contributions to protein activity and fitness.•Hot-spot residues, stability and specificity determinants can be identified. 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.