Conformational changes of proteins are required for nearly all biological functions and inappropriate conformational transitions are associated with numerous pathologies. In particular, processing of DNA and RNA molecules by proteins is fundamental to human health, including cell division and homeostasis, and disease, including cancer and viral infection. Comprehensive experimental information on the essential contributions of intramolecular dynamics to biological functions of proteins is critical for biophysical theories of equilibrium properties, such as heat capacity and thermal stability; for mechanistic interpretations of kinetic processes, such as enzyme catalysis and ligand recognition; and for design of novel proteins and protein ligands, including pharmaceutical agents. These fundamental issues are exemplified by the protein enzyme ribonuclease HI (EC, RNase H), the founding member of a nucleotidyl-transferase superfamily with a conserved structure and mechanism; other family members include transposase, retroviral integrase, Holliday junction resolvase, and RISC nuclease Argonaute. RNase H is distributed widely in prokaryotes and eukaryotes, and HIV retroviral reverse transcriptase contains a C-terminal RNase H domain. RNase H enzymes hydrolyze the RNA strand of DNA/RNA hybrid molecules involved in DNA replication and viral reverse transcription. The goal of the research program is to define the molecular determinants of catalytic activity of RNase H by comparing the structural, dynamical and enzymatic properties of homologous proteins derived from organisms adapted for life in different thermal environments. The extreme temperature dependence of protein conformational properties and activities means that thermal adaptation comprises a natural experiment exploring the linkage between structure, dynamics, and function.
The specific aims for this project are to (i) evaluate the role of the conformational equilibrium of the essential handle loop region in substrate recognition and thermal adaptation, (ii) quantify the importance of population shifts of other substrate-binding loops and amino acid sidechains in modulating affinity, and (iii) explicate the contributions of correlated conformationl dynamics, pre-organization, and conformational rearrangement of active site residues to activity. These objectives are supported by development of improved approaches for characterizing protein dynamics by NMR spectroscopy and MD simulation. This research program will explicate at a level of unprecedented detail molecular aspects of catalysis in this paradigmatic system that are critical for understanding normal and abnormal biological functions of proteins.

Public Health Relevance

Processing of DNA and RNA molecules by proteins is fundamental to human health, including cell division and homeostasis, and disease, including cancer and viral infection. Proteins of the ribonuclease H family of enzymes the subjects of the proposal and are paradigms for understanding aspects of catalysis of DNA/RNA hybrid molecules in replication, reverse transcription, and antisense technologies.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
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Macromolecular Structure and Function B Study Section (MSFB)
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Wehrle, Janna P
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Columbia University (N.Y.)
Schools of Medicine
New York
United States
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Hsu, Andrew; O'Brien, Paul A; Bhattacharya, Shibani et al. (2018) Enhanced spectral density mapping through combined multiple-field deuterium 13CH2D methyl spin relaxation NMR spectroscopy. Methods 138-139:76-84
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O'Connell, Nichole E; Lelli, Katherine; Mann, Richard S et al. (2015) Asparagine deamidation reduces DNA-binding affinity of the Drosophila melanogaster Scr homeodomain. FEBS Lett 589:3237-41

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