Enzyme Dynamics

Researchers have invoked thermal motions within enzyme active sites at femtosecond to picosecond time scales to explain characteristic features of the temperature dependence of primary kinetic isotope effects for enzyme-catalyzed hydrogen transfers. Although some computational studies support a role for these motions, experimental characterization of such fluctuations is lacking. We use 2D IR spectroscopy to characterize the protein dynamics in enzyme active sites and how those dynamics change with different inhibitors bound or as a result of select mutations. We can then correlate the efect of those mutations on the dynamics with the changes in the kinetic isotope effect to understand what role, if any, such motions play in the enzyme-catalyzed hydrogen transfer reaction. 

Read more about the enzymes we study and the approaches we are using for these experiments...

Hydrogen Bonding and Proton Transfer

Hydrogen bonding and proton transfer are at the heart of a wide range of chemical applications including acid-base chemistry, enzyme catalysis, and energy transduction and storage. Vibrational spectroscopy is a sensitive reporter of hydrogen bonding and proton transfer because they induce line broadening, vibrational couplings, and frequency shifts. We use 2DIR spectroscopy to characterize the molecular interactions that underlie these specroscopic features. Specifically, we are preparing a strongly hydrogen-bonded complex that is in equilibrium between the neutral and ion-pair conformations. Using a remote vibration on either the acid or the base we can then follow the chemical exchange between these species at equilibrium.

Read more about the systems we are studying and the interesting spectroscopy that they exhibit...

Developing New Probes for 2D IR

Among the major challeneges in 2D IR spectroscopy is the need to place a spectroscopic chromophore in a position where it is able to sense and report on the desired chemical dynamics. The chromophore must exhibit a unique spectroscopic transition that is distinguishable from other bibrational modes within the sample and exhibit sensitivity to the chemical dynamics of interest. The chromophore should also exert a minimal perturbation on the system. Sometimes these requirements can be satisfied with off-the-shelf, commercially available chromophores, like the azide anion, in many cases, however, finding a good chromophore is more difficult, such as when placing a probe in the active site of an enzyme. We have worked extensively on developing and characterizing chromophores that can be used in a wide range of enzymes to probe their active-site dynamics.

Read more about these chromophores and their applicability to different enzymatic systems...