The basic normal functioning of a cell is the consequence of a delicately balanced regulation of various cellular activities. My laboratory is interested in elucidating the molecular mechanisms of regulation, in particular, the ground rules employed in recognizing specific targets and transmitting of signals among these macromolecular components. There are three biological systems being investigated.
CRP is an ideal system for elucidating the mechanism of adaptation to a change in environment. CRP regulates the expressions of 100 or more genes in E. coli whose survival depends on its ability to express proteins to transport and metabolize food sources which fluctuate. An integrated approach consisting of an integration of thermodynamics of complex formation and protein dynamics is applied to investigate CRP. As a consequence, results of our study led to a major conceptual revelation; namely, a linear correlation between protein dynamics and allosteric parameters. This implies that allosteric behavior tracks the dynamic motion of the CRP molecule. The significance of this relationship is that we have a physical property of CRP as a target, instead of a description, on an ad hoc basis, of the impact of each structural perturbation, as shown in the figure, on the functional energetics. As a consequence, we can integrate the computation and structural approaches to reveal information on protein dynamics and long range connectivity among structural elements.
West Nile virus is a neurotropic flavivirus which cycles between birds and mosquitoes with humans being accidental hosts of infection. The viral envelope protein serves as both a receptor-binding and a fusion protein. Flaviviruses can be transmitted by either mosquitoes or ticks. They are associated with either encephalitic or viscerotropic diseases. Although the atomic structures of the domain 3 of the envelope proteins are very similar, we have shown that the differences in the biophysical properties of these domains correlate with biological properties of these viruses. At present we are investigating the molecular mechanism of neutralization and effect of mutations. (Structure of WNV domain 3 is kindly provided by Dr. David Volk).
The PK system provides a unique opportunity to define the network of pathways involved in the allosteric regulatory of this key glycolytic enzyme. The availability of natural PK mutants of human patients suffering from PK Deficiency has identified some of the key amino acid residues. Previous studies have shown that a single amino acid mutation to the non-allosteric PK at either the Y (S402P) or Z (T340M) subunit interface can confer a certain level of allosteric regulation. In an effort to elucidate the roles of the intersubunit interaction in signal transmission and the functional structural connectivity between these interfaces, the rabbit PK mutant S402P was crystallized and its structure solved to 2.8%. The overall S402P PK structure is nearly identical to the wild type structure within experimental error. However, significant differences in the conformation of the backbone are found at the site of mutation. In addition, there is an unusual face-face interaction between the phenyl rings of Phe 25 of one chain with its counterpart of the chain across the Z interface. At the opposite end of the Z interface, there is a loss of an intersubunit salt bridge between Asp 177 and Arg 341 of the opposing subunit. Concurrent with the loss of the salt-bridge is an increase in the conformational flexibility of one of the domains which contains the active site. Comparison of previous PK structures shows a correlation between an increased movement with the loss of the Asp 177-Arg 341 salt bridge. The crystallographic and solution physical studies yield a consistent set of results to establish a functional linkage between the Y and z interfaces in regulating the interconversion of conformational states of rabbit PK. Additional studies on various mutants are on going. Results of this study will provide the necessary information to address the fundamental issue of the pathway of signal transduction in allosteric systems.
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