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

Tripathi, Arti and Varadarajan, Raghavan (2014) Residue specific contributions to stability and activity inferred from saturation mutagenesis and deep sequencing. In: CURRENT OPINION IN STRUCTURAL BIOLOGY, 24 . pp. 63-71.

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Abstract

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.

Item Type:	Journal Article
Publication:	CURRENT OPINION IN STRUCTURAL BIOLOGY
Publisher:	CURRENT BIOLOGY LTD
Additional Information:	Copyright for this article belongs to the CURRENT BIOLOGY LTD, 84 THEOBALDS RD, LONDON WC1X 8RR, ENGLAND
Department/Centre:	Division of Biological Sciences > Molecular Biophysics Unit
Date Deposited:	11 Jun 2014 10:32
Last Modified:	11 Jun 2014 10:32
URI:	http://eprints.iisc.ac.in/id/eprint/49198

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