Experimental Lab

The following scheme demonstrates the basics of our approach using E. coli’s essential enzyme dihydrofolate reductase (DHFR) as a model protein:

Experimental molecular evolution


The experimental approach. (1) 10 DHFR residues, predominantly from the hydrophobic core and at least 4Å from the cofactor (NADPH) and substrate (DFH) binding sites, were chosen for mutagenesis based on the structural and phylogenetic. (2) 16 single mutants were generated, cloned into pET vector, expressed and purified. (3) Gibbs free energy difference between folded and unfolded states (G), apparent mid-transition temperature of unfolding (Tmapp), and catalytic parameters (kcat, Km) were measured. (4) A site-directed chromosomal mutagenesis method was developed to introduce in vitro characterized mutations into chromosomal folA gene of E. coli’s MG1655 strain without perturbing the gene’s regulatory region. (5) Fitness effects of the introduced mutations were measured by growth competition of mutant strains with wtDHFR strain.

We are currently focusing our efforts on the following topics:

  1. Role of protein homeostasis machinery in shaping the fitness effects of mutations
  2. Co-evolution of expression levels and aggregation propensity of proteins
  3. Understanding the role of protein topology in non-specific protein-protein interactions
  4. Understanding the cons and pros of protein overexpression as an evolutionary strategy to acquire new activity



Selected papers on experimental molecular evolution:

  1. Protein Quality Control Acts on Folding Intermediate to Shape the effects of Mutations on Organismal Fitness
  2. Soluble oligomerization provides a beneficial fitness effect on destabilizing mutations