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. These ground rules are embedded universally in the coupling between two linkage schemes. These schemes include the linkage among multiple thermodynamic parameters governing the biological functions and the intra- and inter-macromolecular networks of communication. There are three biological systems being investigated.
E .coli cAMP Receptor Protein (CRP)
CRP is a well-established allosteric system for elucidating the mechanism of adaptation to a change in environment. The fluctuation of cAMP cellular concentration modulates the ability of CRP to regulate the expressions of 100 or more genes in E.coli. 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 on the functional energetics. As a consequence, we integrate the computation and structural approaches to reveal information on protein dynamics and long range connectivity among structural elements, as shown in the figure. All the sites of mutation reside in the network of connectivity. Thus, we have successfully established a set of basic parameters for in-depth probing the ground rules of allostery.
Mechanism for the Evasion of Antibody Neutralization in Flaviviruses
Infectious diseases caused by flaviviruses are major concerns in the public health community, particularly those that are resistant to antibody-mediated neutralization. The viral envelope protein serves as both a receptor-binding and a fusion protein. Mutations in the epitopes of antigenic proteins can confer viral resistance to antibody-mediated neutralization. We used an ensemble-based algorithm to characterize the effects of mutations on the thermodynamics of protein conformational fluctuations. We determined an intimate relationship between the susceptibility of a residue to thermodynamic perturbations and epitope location. This relationship allows the successful identification of the primary epitopes in each ED3, despite their high sequence and structural similarity. Mutations that allow the ED3 to evade detection by the antibody either increase or decrease conformational fluctuations of the epitopes through local effects or long-range interactions. Spatially distant interactions originate in the redistribution of conformations of the ED3 ensembles, not through a mechanically connected array of contiguous amino acids. These results reconcile previous observations of evasion of neutralization by mutations at a distance from the epitopes. Finally, we established a quantitative correlation between subtle changes in the conformational fluctuations of the epitope and large defects in antibody binding affinity. This correlation suggests that mutations that allow viral growth, while reducing neutralization, do not generate significant structural changes and underscores the importance of protein fluctuations and long-range interactions in the mechanism of antibody-mediated neutralization resistance.
Mammalian Pyruvate Kinase
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. Mammalian pyruvate kinase exists in four isoforms with characteristics tuned to specific metabolic requirements of different tissues. All of the isoforms, except the muscle isoform, exhibit typical allosteric behavior. The case of the muscle isoform is a conundrum. It is inhibited by an allosteric inhibitor, Phe, yet it has traditionally not been considered as an allosteric enzyme. In this series of study, an energetic landscape of rabbit muscle pyruvate kinase (RMPK) was established. The phenomenon of inhibition by Phe is shown to be physiological. Furthermore, the thermodynamics for the temperature fluctuation and concomitant pH change as a consequence of muscle activity were elucidated. We have shown that (1) the differential number of protons released or absorbed with regard to the various linked reactions adds another level of control to shift the binding constants and equilibrium of active/inactive state changes (the latter controls quantitatively the activity of RMPK); (2) ADP plays a major role in the allosteric mechanism in RMPK under physiological temperatures (depending on the temperature, ADP can assume dual and opposite roles of being an inhibitor by binding preferentially to the inactive form and a substrate); and (3) simulation of the RMPK behavior under physiological conditions shows that the net results of the 21 thermodynamic parameters involved in the regulation are well-tuned to allow the maximal response of the enzyme to even minute changes in temperature and ligand concentration.