Event

Using Physical Chemistry to Solve Important Problems in Molecular Biology and Nanotechnology

In this seminar I will present two examples of very recent work on applying methods of physical chemistry to address important problems in molecular biology and nanotechnology. The first is the classical conundrum in molecular biology of how transcription factors efficiently find their unique target sequence within the enormous pool of alternative binding sites found on genomic DNA. Investigating interactions between the DNA-binding domain from engrailed and DNA molecules of varying size with single-molecule fluorescence methods and statistical mechanical modeling we have discovered an entropic mechanism that provides an elegant solution to this long-standing puzzle. The mechanism stems from our finding that transcription factors bind with relatively low affinity to repetitive clusters of degenerate target sequences found in the regulatory regions of the relevant gene. The large numbers of degenerate binding sites act as an amplifying antenna that increases the local concentration of the transcription factor by over 100-fold, thus enabling extremely quick homing and sub-nanomolar apparent affinities while maintaining the relatively fast off-rates required for nimble switching of gene expression. This mechanism also suggests a novel strategy to control gene expression levels.

The second example is the creation of protein assemblies with defined symmetry and stoichiometry, a major goal of nanotechnology. Recent successes have centered on static assemblies. Our goal is to build assemblies that can be controlled externally. To this end we devised a strategy in which assembly is thermodynamically coupled to partial unfolding of the monomer protein. In this scheme the folded protein remains monomeric, but a partial unfolding event controlled by either an external (temperature, mechanical force, photochemical) or internal (mutations, binding partner) variable converts the monomer into an assembly-competent state that forms the assembly. As proof of concept we engineered chymotrypsin inhibitor 2 (CI2), a very stable, monomeric two-state folding protein that crystallizes forming a dodecameric ring structure, to introduce the trigger mechanism and stabilize the ring assembly. Using a battery of physical chemical techniques (kinetics, analytical ultracentrifugation, light scattering, nuclear magnetic resonance, X-ray crystallography, electron microscopy, differential scanning calorimetry and computational modeling) we could demonstrate the feasibility of this approach and its potential to be generalizable.