In Vivo Pathways of Molecular Evolution: Acquisition of Thermostability by Mesophilic Adenylate Kinase

William Marsh Rice University, Houston TX

Investigators

Abstract

Using in vivo molecular evolution the P.I. is studying how an organism mutates an essential gene as a response to changes in its environment. The model organism is a genetically modified strain of B. stearothermophilus (a thermophile) that has had the wild type copy of the essential adenylate kinase gene (adk) replaced by the mesophilic B. subtilis gene. The grant focuses on two key attributes of molecular evolution; firstly, what are the characteristics of bacterial populations as they adapt to environmental extremes, and secondly, what are the physicochemical properties of the proteins generated from these selections. The experiments follow populations of mutant bacteria as they compete for survival and will use x-ray crystallography to examine the stereochemical basis for increases in thermostability. Sequence and structure based comparisons of mesophilic and thermophilic homologs have suggested that numerous changes in amino acid sequence can increase thermostability, whereas in vitro directed evolution has shown that a smaller number of changes can frequently bring about the required changes in activity/thermostability. One conclusion frequently drawn from these observations is that many of the differences seen in sequence are the result of evolutionary drift, rather than evolutionary necessity. To determine the principles that guide evolution of a protein, the mutational path(s) taken by a population of bacteria as they evolve an essential protein to higher thermostability is being determined. Together with atomic analysis of the protein intermediates along this path, the complex sequence surface that constitutes the mutational path to thermostability for the model protein will be explored. Altering conditions of the evolution experiment can also change the "mutational path" used by the bacterial populations and allow further insights into evolution. Thus the primary goal of the work is to elucidate the pathway(s) used in vivo and to apply biophysical methods to understand the physical basis for the elaborated mutational pathway(s).