We study the mechanistic chemistry of enzymes and ion channels. One area of enzymology we are investigating is how enzymes use the free energy of ATP hydrolysis to perform work. We are also interested in the kinetic and mechanistic aspects of interactions between enzymes and ion channels, particularly as they relate to physiology. In the area of ion channels, we would like to understand the conformational changes that convert channels from closed (i.e., ion-impermeable) states to open (i.e., ion-permeable) states. In the long term, we hope that these research projects will provide not only insight into the mechanisms of molecular machines and protein conformational changes but also useful information for areas of science outside of mechanistic biochemistry, such as the design of molecule-scale devices and the study of genetic and metabolic networks.

The ClpA/ClpP ATP-dependent protease is one example of an enzyme that hydrolyzes ATP in order to do work on a substrate. The ClpA hexamer of this enzyme complex hydrolyzes ATP and uses the free energy of hydrolysis to unfold protein substrates. We are initiating kinetic studies to address questions such as whether the unfolding takes place as a global denaturation or as a gradual unraveling. We are also interested in the question of whether ClpP, which forms a chamber of seven protease active sites, accomplishes its remarkably efficient proteolysis by shuttling the unfolded protein between active sites in the chamber.

The free energy of ATP hydrolysis can also fuel protein conformational changes that are more subtle than protein unfolding. In the ATP-sensitive potassium channel, the potassium channel Kir 6.2 is associated with the sulfonylurea receptor (SUR), an enzyme which hydrolyzes ATP and is homologous to a family of ATP-dependent transporters. Recent work suggests that ATP hydrolysis by SUR drives the channel through a cycle of open and closed conformational states. To test this hypothesis, we will use single molecule kinetic methods to determine whether these conformational changes violate microscopic reversibility, as would be expected if they are coupled to irreversible ATP hydrolysis. The reactivity of engineered cysteines will be used to investigate which domains of the channel might serve as moving parts in the ATP-driven macromolecular machine.

In the nervous system, protein conformational changes serve to sense electrical signals. The mechanism by which transmembrane voltage shifts the equilibrium between the open and closed conformations of voltage-sensitive potassium channels has been the subject of intense investigation. One open mechanistic question is how motion of the channel’s voltage sensing region is coupled to motions of the pore region. We will study a mutant channel in which binding of cadmium to cysteine and histidine residues in the pore region locks the channel into the open state. The effect that this structural perturbation at the pore has on charge motion at the voltage sensor will depend on the coupling of conformational changes in the two regions. A related goal is the engineering of binding sites for redox-active metals in Shaker, with the goal of studying electron transfer at the single-molecule level.

S. Licht, A. Sonnleitner, S. Weiss, P.G. Schultz (2003) A Rugged Energy Landscape Mechanism for Trapping of Transmembrane Receptors During Endocytosis, Biochemistry, accepted for publication.

S. Licht, C. Lawrence, J. Stubbe (1999) Class II Ribonucleotide Reductases Catalyze Carbon-Cobalt Bond Reformation on Every Turnover, Journal of the American Chemical Society, 121, 7463-7468.

S. Licht, G.J. Gerfen, J. Stubbe (1996) Thiyl Radicals in Ribonucleotide Reductases, Science 271, 477-481.