Pipetting in style.

Matthew Peña  

Summary of Research:

Changes in protein structure at the atomic level are the basis for protein evolution and are critical for adaptation. Continuous evolution of bacterial populations can be used to determine mutational pathways and adaptive mechanisms of increased fitness during natural selection. A more precise understanding of the biophysical origins of fitness is central to predicting evolutionary outcomes.

In the 'weak link' approach, a functional and essential gene within the chromosome of the organism is replaced by a maladapted ortholog. A weak link was created by replacing the native adenylate kinase (adk) gene of Geobacillus stearothermophilus, a thermophile, with that of Bacillus subtilis, a mesophile, to produce the strain NUB3621-R:ThEV. Adenylate kinase is essential to adenylate homeostasis within the cell. Introducing the weak link into G. stearothermophilus produces a temperature sensitive phenotype unable to survive at higher temperatures that would otherwise be habitable. Within a population undergoing selection for growth at higher non-permissive temperatures, mutations in adk facilitate growth by increasing enzyme stability and shifting activity. The initial mutant, Q199R, rose to fixation and served as the background for all double-mutants that followed.

In vitro biophysical analysis of the five double-mutant enzymes correlates to in vivo fitness. Folding studies show a general trend of decreased unfolding rate compared to Q199R, with the exception of A193V, which also exhibits increased folding rate.  Activity assays that monitor ADP production reveal that higher activity at specific temperatures correlates with the success of the strains at their respective temperatures.  Folding dynamics, including a mutant's propensity towards aggregation, certainly contribute to organismal fitness, but it remains to be seen how these values will correlate to a mutants success.  The activity profiles correspond so closely to the success of the mutants over the range of temperatures that activity may serve as a proxy for fitness. Crystal structures of the mutant enzymes reflect a diverse set of mechanisms by which stability is increased and activity is maintained. Together, these mutants illustrate both great and subtle adaptations produced through the smallest unit of evolutionary change.

My work involves the analysis of enzymatic rates to provide a complete description of the kinetic properties of adenylate kinase mutants.  I then use information describing folding dynamics and activity to form fitness functions to be used in simulations of evolution in an asexual population consisting of these mutants.

Documents available:

Keck Center Annual Research Conference 2008 Poster

Publications:

Couñago, R., Wilson C.J., Peña, M.I., Wittung-Stafshede, P., Shamoo, Y. (2008).  An adaptive mutation in adenylate kinase that increases organismal fitness is linked to stability-activity trade-offs.  Prot. Eng. Des. Sel. 21, 19-27.