Dr. Stephan Witt's Research Focus

Major Research Interests:
Structure, mechanism and regulation of 70-kDa molecular chaperones; misfolded proteins, chaperones, and human disease. Alpha synuclein and Parkinson’s disease.

One area of research in my lab focuses on the mechanism of action of 70-kDa molecular chaperones, which are also called heat shock-70 proteins (hsp70). The genes that encode 70-kDa chaperones are expressed in nearly all organisms, and the genes have been highly conserved through evolution. Constitutively expressed 70-kDa chaperones participate in reactions that are common to all cells, such as, protein folding, transport, and assembly. The expression of other 70-kDa chaperones is induced by stresses such as heat shock, ethanol, heavy metals, organic solvents, and even some drugs. Inducible 70-kDa chaperones are thought to prevent and even reverse the aggregation of other proteins. The common mechanistic feature of the reaction of 70-kDa chaperones with its substrates is that the chaperone protein utilizes free energy from ATP binding and hydrolysis to reversibly switch between high- and low-affinity conformations. The high-affinity state, which has ADP bound in the ATPase domain of the chaperone, tightly binds substrate, whereas the low-affinity state, which has ATP bound in the ATPase domain of the chaperone, weakly binds substrate. This reversible, high- to low-affinity transition is like a molecular switch. Using a variety of techniques from biophysical chemistry and molecular biology, we are probing the kinetics, mechanism and regulation (by the two cochaperones, DnaJ and GrpE) of this molecular switch in the 70-kDa chaperone expressed by Escherichia coli, DnaK. This research is funded by the National Institutes of Health.

Another area of research in my lab focuses on bacterial nucleotide exchange factors, such as GrpE. GrpE catalyzes the release of ADP from DnaK. Interestingly, Yeast and other eukaryotes also express GrpE-like nucleotide exchange factors, which are typically located in the mitochondria. Several aspects of GrpE biology and biochemistry are underway in my laboratory. In one project, we are exploring how mutations in the gene MGE1, a yeast gene that encodes a GrpE-like protein, affect mitochondrial function. In another project, we are using surface plasmon resonance to study how mutations in the GrpE protein affect the kinetics of GrpE binding and release from DnaK. Our goal is to understand why GrpE-like exchange factors seem to be needed specifically in mitochondria.

Another area of research involves using yeast as a model organism to study the mechanism of alpha synuclein ( Syn) toxicity. Alpha synuclein, one of the most abundant proteins expressed in human neurons, has been implicated in Parkinson’s disease. From expressing green fluorescent protein fusions of human wild type Syn and two mutants (GFP-WT, GFP-A53T, and GFP-A30P) in yeast, we have discovered that GFP-WT and GFP-A53T is localized to the inner plasma membrane and that GFP-A30P is distributed through out the cytosol. Each of these Syns inhibits cell growth. Experiments are currently underway to deduce the mechanism of Syn toxicity.