Predictive Shifts in Free Energy Couple Mutations to Their Phenotypic Consequences.

Griffin Chure, Manuel Razo-Mejia, Nathan M. Belliveau, Tal Einav, Zofii A. Kaczmarek, Stephanie L. Barnes, Mitchell Lewis, Rob Phillips
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Scientific Abstract

Mutation is a critical mechanism by which evolution explores the functional landscape of proteins. Despite our ability to experimen- tally inflict mutations at will, it remains difficult to link sequence- level perturbations to systems-level responses. Here, we present a framework centered on measuring changes in the free energy of the system to link individual mutations in an allosteric transcrip- tional repressor to the parameters which govern its response. We find that the energetic effects of the mutations can be categorized into several classes which have characteristic curves as a func- tion of the inducer concentration. We experimentally test these diagnostic predictions using the well-characterized LacI repressor of Escherichia coli, probing several mutations in the DNA bind- ing and inducer binding domains. We find that the change in gene expression due to a point mutation can be captured by modifying only the model parameters that describe the respec- tive domain of the wild-type protein. These parameters appear to be insulated, with mutations in the DNA binding domain altering only the DNA affinity and those in the inducer binding domain altering only the allosteric parameters. Changing these subsets of parameters tunes the free energy of the system in a way that is concordant with theoretical expectations. Finally, we show that the induction profiles and resulting free energies associated with pairwise double mutants can be predicted with quantitative accu- racy given knowledge of the single mutants, providing an avenue for identifying and quantifying epistatic interactions.