Mutagenesis-based definitions and probes of residue burial in proteins

Abstract

Every residue of the 101-aa Escherichia coli toxin CcdB was substituted with Ala, Asp, Glu, Lys, and Arg by using site-directed mutagenesis. The activity of each mutant in vivo was characterized as a function of Controller of Cell Division or Death B protein (CcdB) transcriptional level. The mutation data suggest that an accessibility value of 5% is an appropriate cutoff for definition of buried residues. At all buried positions, introduction of Asp results in an inactive phenotype at all CcdB transcriptional levels. The average amount of destabilization upon substitution at buried positions decreases in the order Asp>Glu>Lys>Arg>Ala. Asp substitutions at buried sites in two other proteins, maltose-binding protein and thioredoxin, also were shown to be severely destabilizing. Ala and Asp scanning mutagenesis, in combination with dose-dependent expression phenotypes, was shown to yield important information on protein structure and activity. These results also suggest that such scanning mutagenesis data can be used to rank order sequence alignments and their corresponding homology models, as well as to distinguish between correct and incorrect structural alignments. With continuous reductions in oligonucleotide costs and increasingly efficient site-directed mutagenesis procedures, comprehensive scanning mutagenesis experiments for small proteins/domains are quite feasible.

Fig. 1.

Ninety-six-well screening to obtain phenotypes of CcdB mutants as a function of inducer arabinose concentration at 37°C. Mutants were transformed into E. coli strain Top10 and plated at 0% (A), 0.001% (B), 0.01% (C), and 0.1% (D) arabinose. Identical grid positions on different plates correspond to the same mutant. Cells transformed with active mutants do not grow, whereas those transformed with inactive mutants will grow. The fraction of mutants showing an active phenotype clearly increases with increasing inducer concentration.