Cognate mutants in orthologous proteins: the Eliza Doolittle experiment. prof Daniel L. Hartl, Harvard University, USA

Increasing evidence supports the hypothesis that most amino acid polymorphisms within populations are slightly deleterious. The polymorphisms tend to be surface residues accessible to solvent. Slightly adverse effects on protein folding, stability, aggregation, or degradation may mediate these deleterious effects. Compensatory mutations could therefore account for the observation that, unlike polymorphisms within populations, most amino acid replacements between related species are subject to weak positive selection. One test of this model is to examine cognate amino acid replacements in orthologous proteins. If fixed differences affect protein folding, stability, aggregation, or degradation, then cognate replacements should in some cases have different effects on protein stability either quantitatively or even qualitatively. We have carried out experimental tests of bacterial DHFR orthologs carrying cognate mutations conferring resistance to trimethoprim in transgenic E. coli. Our results support a model in which cognate mutations in orthologous proteins can result in major differences in protein stability. The cognate mutants also significantly extend the dynamic range of biophysical parameters of DHFR, enabling a comprehensive biophysical mapping of the fitness landscape. We show that kinetic flux theory provides an accurate model to predict quantitatively the resistance phenotypes at different trimethoprim concentrations from the explicit parameterization of a combination of molecular and protein-abundance data. Taking into account the protein quality control mechanism that govern the intracellular abundance of DHFR, drug resistance can be predicted solely from molecular biophysical properties of DHFR. The results provide a comprehensive genotype-phenotype map for DHFR and demonstrate the need for accurate prediction of the effects of mutations on molecular properties of target enzymes.