title: Molecular Determinants of Mutant Phenotypes, Inferred from Saturation Mutagenesis Data.
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 (PSD95pdz3) 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 on mutant 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.
authors: Molecular Biophysics Unit, Indian Institute of Science, Bangalore, India.
authors: Arti
authors: A
authors: Tripathi
authors: Molecular Biophysics Unit, Indian Institute of Science, Bangalore, India.
authors: Kritika
authors: K
authors: Gupta
authors: Molecular Biophysics Unit, Indian Institute of Science, Bangalore, India.
authors: Shruti
authors: S
authors: Khare
authors: Molecular Biophysics Unit, Indian Institute of Science, Bangalore, India.
authors: Pankaj C
authors: PC
authors: Jain
authors: Molecular Biophysics Unit, Indian Institute of Science, Bangalore, India.
authors: Siddharth
authors: S
authors: Patel
authors: Molecular Biophysics Unit, Indian Institute of Science, Bangalore, India.
authors: Prasanth
authors: P
authors: Kumar
authors: Molecular Biophysics Unit, Indian Institute of Science, Bangalore, India.
authors: Ajai J
authors: AJ
authors: Pulianmackal
authors: Molecular Biophysics Unit, Indian Institute of Science, Bangalore, India.
authors: Nilesh
authors: N
authors: Aghera
authors: Molecular Biophysics Unit, Indian Institute of Science, Bangalore, India Jawaharlal Nehru Center for Advanced Scientific Research, Bangalore, India varadar@mbu.iisc.ernet.in.
authors: Raghavan
authors: R
authors: Varadarajan
mesh_terms: D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D020134:Catalytic Domain; D004926:Escherichia coli / Q000235:genetics*; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods; D008969:Molecular Sequence Data; D016296:Mutagenesis; D016297:Mutagenesis, Site-Directed / Q000379:methods; D009154:Mutation; D010641:Phenotype; D011487:Protein Conformation; D055550:Protein Stability; D011506:Proteins / Q000235:genetics; D020539:Sequence Analysis, Protein; D013329:Structure-Activity Relationship
chemical_list: D011506:Proteins
keywords: deep sequencing; fitness effect prediction; mutagenesis; protein folding