Molecular Determinants of Mutant Phenotypes, Inferred from Saturation Mutagenesis Data

Tripathi, Arti and Gupta, Kritika and Khare, Shruti and Jain, Pankaj C and Patel, Siddharth and Kumar, Prasanth and Pulianmackal, Ajai J and Aghera, Nilesh and Varadarajan, Raghavan (2016) Molecular Determinants of Mutant Phenotypes, Inferred from Saturation Mutagenesis Data. In: MOLECULAR BIOLOGY AND EVOLUTION, 33 (11). pp. 2960-2975.

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Abstract

Understanding how mutations affect protein activity and organismal fitness is a major challenge. We used saturation mutagenesis combined with deep sequencing to determine mutational sensitivity scores for 1,664 single-site mutants of the 101 residue Escherichia coli cytotoxin, CcdB at seven different expression levels. Active-site residues could be distinguished from buried ones, based on their differential tolerance to aliphatic and charged amino acid substitutions. At nonactive-site positions, the average mutational tolerance correlated better with depth from the protein surface than with accessibility. Remarkably, similar results were observed for two other small proteins, PDZ domain (PSD95 pdz3) and IgG-binding domain of protein G (GB1). Mutational sensitivity data obtained with CcdB were used to derive a procedure for predicting functional effects of mutations. Results compared favorably with those of two widely used computational predictors. In vitro characterization of 80 single, nonactive-site mutants of CcdB showed that activity in vivo correlates moderately with thermal stability and solubility. The inability to refold reversibly, as well as a decreased folding rate in vitro, is associated with decreased activity in vivo. Upon probing the effect of modulating expression of various proteases and chaperones onmutant phenotypes, most deleterious mutants showed an increased in vivo activity and solubility only upon over-expression of either Trigger factor or SecB ATP-independent chaperones. Collectively, these data suggest that folding kinetics rather than protein stability is the primary determinant of activity in vivo. This study enhances our understanding of how mutations affect phenotype, as well as the ability to predict fitness effects of point mutations.

Item Type:	Journal Article
Publication:	MOLECULAR BIOLOGY AND EVOLUTION
Additional Information:	copy right for this article belongs to the OXFORD UNIV PRESS, GREAT CLARENDON ST, OXFORD OX2 6DP, ENGLAND
Department/Centre:	Division of Biological Sciences > Molecular Biophysics Unit
Date Deposited:	03 Dec 2016 10:09
Last Modified:	03 Dec 2016 10:09
URI:	http://eprints.iisc.ac.in/id/eprint/55404

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