Connecting Protein Conformation and Dynamics with Ligand-Receptor Binding Using Three-Color Förster Resonance Energy Transfer Tracking.

Specific binding between biomolecules, i.e., molecular recognition, controls virtually all biological processes including the interactions between cells and biointerfaces, both natural and synthetic. Such binding often relies on the conformation of biomacromolecules, which can be highly heterogeneous and sensitive to environmental perturbations, and therefore difficult to characterize and control. An approach is demonstrated here that directly connects the binding kinetics and stability of the protein receptor integrin αvß3 to the conformation of the ligand fibronectin (FN), which are believed to control cellular mechanosensing. Specifically, we investigated the influence of surface-adsorbed FN structure and dynamics on αvß3 binding using high-throughput single-molecule three-color Förster resonance energy transfer (FRET) tracking methods. By controlling FN structure and dynamics through tuning surface chemistry, we found that as the conformational and translational dynamics of FN increased, the rate of binding, particularly to folded FN, and stability of the bound FN-αvß3 complex decreased significantly. These findings highlight the importance of the conformational plasticity and accessibility of the arginine-glycine-aspartic acid (RGD) binding site in FN, which, in turn, mediates cell signaling in physiological and synthetic environments.

A Synonymous Mutation Upstream of the Gene Encoding a Weak-Link Enzyme Causes an Ultrasensitive Response in Growth Rate.

UNLABELLED: When microbes are faced with an environmental challenge or opportunity, preexisting enzymes with promiscuous secondary activities can be recruited to provide newly important functions. Mutations that increase the efficiency of a new activity often compromise the original activity, resulting in an inefficient bifunctional enzyme. We have investigated the mechanisms by which growth of Escherichia coli can be improved when fitness is limited by such an enzyme, E383A ProA (ProA*). ProA* can serve the functions of both ProA (required for synthesis of proline) and ArgC (required for synthesis of arginine), albeit poorly. We identified four genetic changes that improve the growth rate by up to 6.2-fold. Two point mutations in the promoter of the proBA* operon increase expression of the entire operon. Massive amplification of a genomic segment around the proBA* operon also increases expression of the entire operon. Finally, a synonymous point mutation in the coding region of proB creates a new promoter for proA* This synonymous mutation increases the level of ProA* by 2-fold but increases the growth rate by 5-fold, an ultrasensitive response likely arising from competition between two substrates for the active site of the inefficient bifunctional ProA*. IMPORTANCE: The high-impact synonymous mutation we discovered in proB is remarkable for two reasons. First, most polar effects documented in the literature are detrimental. This finding demonstrates that polar effect mutations can have strongly beneficial effects, especially when an organism is facing a difficult environmental challenge for which it is poorly adapted. Furthermore, the consequence of the synonymous mutation in proB is a 2-fold increase in the level of ProA* but a disproportionately large 5.1-fold increase in growth rate. While ultrasensitive responses are often found in signaling networks and genetic circuits, an ultrasensitive response to an adaptive mutation has not been previously reported.

Differential effects of a mutation on the normal and promiscuous activities of orthologs: implications for natural and directed evolution.

Neutral drift occurring over millions or billions of years results in substantial sequence divergence among enzymes that catalyze the same reaction. Although natural selection maintains the primary activity of orthologous enzymes, there is, by definition, no selective pressure to maintain physiologically irrelevant promiscuous activities. Thus, the levels and the evolvabilities of promiscuous activities may vary among orthologous enzymes. Consistent with this expectation, we have found that the levels of a promiscuous activity in nine gamma-glutamyl phosphate reductase (ProA) orthologs vary by about 50-fold. Remarkably, a single amino acid change from Glu to Ala near the active site appeared to be critical for improvement of the promiscuous activity in every ortholog. The effects of this change varied dramatically. The improvement in the promiscuous activity varied from 50- to 770-fold, and, importantly, was not correlated with the initial level of the promiscuous activity. The decrease in the original activity varied from 190- to 2,100-fold. These results suggest that evolution of a novel enzyme may be possible in some microbes, but not in others. Further, these results underscore the importance of using multiple orthologs as starting points for directed evolution of novel enzyme activities.