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.

Contribution of the Chromosomal ccdAB Operon to Bacterial Drug Tolerance

“…A previous study of E. coli O157:H7 suggests that CcdB O157 is also active and that when (12), were aligned with the CcdB F sequence using the Clustal Omega multiple-sequence alignment tool. Residues that are fully conserved either are active-site residues or form a part of the hydrophobic core (34). (B) CcdB F -gyrase A14 bound crystal structure (PDB no.…”

Systematic Identification of Machine-Learning Models Aimed to Classify Critical Residues for Protein Function from Protein Structure

“…In an attempt to propose a solution to this limitation in the definition of critical residues, here we define a criticality index function ( CI , see Methods); such index encompasses five different ways to account for amino acid replacements (polarity, secondary structure, molecular volume, codon diversity, and electrostatic charge) and consequently provides a quantitative estimate of how tolerant is a given position to mutants. It is important to note that previous methods have been reported to predict mutations that negatively affect protein function [ 23 , 24 , 25 ]; such approaches differ from our approach since our goal is to define which residues are critical rather than to identify which mutations may alter protein function. Our results show that CI is learnable by both centralities and ProtDCal descriptors, and that CI captures different definitions of critical residues tested in this work.…”

Facile measurement of protein stability and folding kinetics using a nano differential scanning fluorimeter

“…The pellet was resuspended in HEG resuspension buffer pH 7.4 (10 mM HEPES, 50 mM EDTA, 10% glycerol containing 10 mM PMSF) and sonicated, followed by centrifugation at 25,000 g, 30 min, 4 °C. The supernatant was incubated with Affi‐Gel15 (Biorad) coupled to CcdA peptide (residues 46–72) and incubated overnight at 4 °C as described previously . The unbound fraction was removed and washed with five times the bed volume of coupling buffer pH 8.3 (0.05 M sodium bicarbonate, 0.5 M sodium chloride).…”

“…Unfolding kinetic traces of fluorescence intensity in 3 M GdnCl as a function of time for CcdB in 10 mM or 200 mM HEPES, pH 8.4 were normalized from 0 to 1 between native and denatured baseline at 3 M GdnCl. The data was analyzed using SigmaPlot™ version 12.5 for Windows™ scientific graphing software, from Systat Software, Inc., San Jose, CA, USA, (http://www.systatsoftware.com), and plots were fitted to a 5 parameter equation for exponential decay for refolding (y = y 0 + a*exp(−bx) + c*exp(−dx)), yielding slow and fast phase rate constants and a 3 parameter exponential rise for unfolding (y = y 0 + a*exp[b*x]) as described previously, where x is the time (in seconds) of refolding/unfolding …”

“…CcdB was expressed under control of the arabinose promoter P BAD in the pBAD24 vector using the CcdB resistant Top10Gyrase strain of E. coli, and purified using affinity chromatography as described previously. 27 A single colony was inoculated into LB medium (HiMedia) and grown at 37 C overnight. Five hundred milliliters of LB medium was inoculated with 1% of the primary inoculum and grown at 37 C until the OD 600 reached 0.6.…”