Probing dynamics within an enzyme family.
EMSL Project ID
28396
Abstract
Enzyme motions associated with catalysis have been found to occur even in the absence of substrates and localize to similar regions. This inherent flexibility may allow important conformations to be dynamically sampled, such as those that initiate substrate binding, suggesting that enzyme dynamics are critical for function. However, it is unknown whether inherent motions are unique to these particular members or are conserved across enzyme families and how these motions are related to sequence, structure, and function. Thus, the long-term goal of this proposed study is to determine how inherent motions compare within an enzyme family and to begin determining the relationship between sequence, structure, dynamics, and function. Such knowledge would be instrumental in understanding the evolutionary pressures that dictate protein motions in addition to those that dictate catalysis. Moreover, structure has aided the development of approaches for the rational design of therapeutics and understanding the dynamics of such moving targets may further advance such strategies.NMR solution studies will be used to probe enzyme motions at atomic resolution. The novelty in this approach is that active enzyme complexes can be monitored because of the nature of the reversible reaction in which they catalyze. This provides an important link between dynamics and function, since how inherently dynamic regions of enzymes are preempted for catalysis can be directly probed, as opposed to inferred from static structures. By combining these methods with mutagenesis, local contributions to the global dynamics within a particular enzyme can be measured and by studying several enzymes within a particular family, the dynamic contributions of both conserved and non-conserved residues can be determined.
Cyclophilins catalyze the reversible cistrans interconversion of peptidyl-prolyl bonds and are both overexpressed in multiple cancers and utilized by several viruses for infection, including Human Immunodeficiency virus type-1 (HIV-1). We have detected motions within the prototypical family member, cyclophilin-A (CypA), in the absence of substrate that are similar to its rate of catalytic turnover. Here, these inherent motions will be characterized to probe whether CypA undergoes similar conformational rearrangements as catalysis using our combination of mutagenesis and NMR. Both enzyme and substrate will be monitored during turnover to identify residue motions associated with catalysis. Finally, these investigations will be extended to several other human cyclophilin family members to determine how motions have been conserved and how amino acid differences may regulate dynamics and, importantly, function.
Project Details
Project type
Limited Scope
Start Date
2008-02-27
End Date
2008-04-23
Status
Closed
Released Data Link
Team
Principal Investigator