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Thermal adaptation of conformational dynamics in ribonuclease H

Stafford, Kate

Structural changes are critical to the ability of proteins, particularly enzymes, to carry out their biological function. However, flexibility also leaves proteins vulnerable to denaturation and degradation; thus a balance must be struck between the dynamics required for function and the rigidity required for maintaining a globular protein's characteristic folded structure. These relationships have been studied in detail through comparison of homologous proteins from organisms adapted to varying properties of the bulk environment. In particular, organisms adapted to temperature extremes offer fruitful platforms for the investigation of adaptive changes in protein stability as a function of environmental pressures. Thermostable proteins are widely reported to be more rigid than their homologs from mesophilic organisms, and those from psychrophiles more flexible; this suggests the possibility of evolutionary conservation of the balance between dynamics and stability. Thus specifically functional aspects of protein dynamics may be isolable through the comparative analysis of members of protein families from organisms adapted to different thermal environments. The best experimental tool for characterizing internal conformational dynamics of proteins on a range of timescales and at site-specific resolution is nuclear magnetic resonance (NMR) spectroscopy, which has found widespread use in the study of protein flexibility and dynamics. However, it is often difficult to provide a detailed structural interpretation of NMR observations. This gap can be bridged using molecular dynamics (MD) simulations, which can directly simulate motional processes that have been observed experimentally. The potential for deep synergy between these two complementary tools has been recognized since MD methods were first applied to biological macromolecules, and recent technological developments have reinforced the mutually beneficial relationship between the two techniques. Ribonuclease HI (RNase H), an 18 kD globular protein that hydrolyzes the RNA strand of RNA:DNA hybrid substrates, has been extensively studied by NMR to characterize the differences in dynamics between homologs from the mesophilic organism \textit{E. coli} and the thermophilic organism \textit{T. thermophilus}. However, these dynamic differences are subtle and difficult to interpret structurally. The series of studies described in the present work was conceived in the pursuit of an improved understanding of the complex relationships between protein dynamics, activity, and thermostability in the RNase H protein family. The organizing principle of the work presented herein has been the close coupling between molecular dynamics simulations and NMR observations, permitting both validation of the MD trajectories by rigorous comparison to experiment and improved interpretation of the dynamics observed by NMR. Previous NMR observations of E. coli and T. thermophilus are integrated into an interpretive framework derived from simulations of the larger RNase H family. First, comparative analysis of molecular dynamics simulations of a total of five homologous RNase H families from organisms of varying preferred growth temperature reveals systematic differences in the conformational dynamics of the handle region, a loop previously identified as contributing to substrate binding. Second, analysis of the effects of activating mutations on the dynamics of ttRNH identifies rotamer dynamics whose contributions to increased catalytic activity can be rationalized in the context of observed differences in sidechain orientation in the wild-type ecRNH and ttRNH simulations. Third, a combined MD-NMR study finds that the active site residues of ecRNH, and likely of the entire RNase H family, are rigid on the ps-ns timescale while undergoing substantial conformational exchange upon Mg2+ binding; this suggests that the active site is electrostatically preorganized for binding the first metal ion, which in turn induces dynamic reorganization at longer timescales. Finally, long-timescale simulations of the RNase H family, despite unexpected local unfolding for some family members, identify handle-loop and rotamer preferences for the C. tepidum RNase H (ctRNH) homolog that unexpectedly differ from those observed for ecRNH and ttRNH, and which can be experimentally tested by NMR spectroscopy of this recently characterized and less well-studied example of an RNase H homolog from a thermophilic organism.



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More About This Work

Academic Units
Biochemistry and Molecular Biophysics
Thesis Advisors
Palmer, Arthur
Ph.D., Columbia University
Published Here
November 7, 2013