Conformational changes of proteins are required for nearly all proper biological functions and inappropriate conformational changes 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. This research project will use multidimensional NMR spectroscopy to address these fundamental issues. One long-term goal is to define the molecular determinants of stability and catalytic activity of the enzyme ribonuclease HI (RNase H) by comparing the structural, dynamical and enzymatic properties of homologous proteins derived from Escherichia coli and the extremely thermophilic bacterium Thermus thermophilus. The enzyme hydrolyzes the RNA strand of DNA/RNA hybrid molecules involved in DNA replication, viral reverse transcription, and antisense technology. RNase H is distributed widely in prokaryotes and eukaryotes, and HIV retroviral reverse transcriptase contains a C-terminal RNase H domain.
The specific aims for this project are to determine the molecular determinants of the difference in catalytic activity of the two homologous enzymes at moderate temperatures and to determine how necessary conformational rigidity is maintained by the thermophilic protein at elevated temperatures. Another long-term goal is to define the molecular determinants of ligand binding, including aspects of specificity, in DNA recognition by the yeast protein GCN4. Motifs that recognize specific DNA sequences are ubiquitous components of proteins that regulate gene expression. GCN4 is the prototypical member of the bZip family of transcription activators and represents an example of induced fit molecular recognition through a disorder-order transition associated with DNA binding.
The specific aim for this project is to evaluate the role of transient local structures in the molecular recognition mechanism. These objectives are supported by a specific aim to develop improved approaches for characterizing protein dynamics by NMR spectroscopy and computer simulation. This research will explicate molecular aspects of catalysis and recognition in these two paradigmatic systems that are critical for understanding normal and abnormal biological functions.

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 and the bZip family of transcription factors are the subjects of the proposal and are paradigms for understanding aspects of catalysis of DNA/RNA hybrid molecules in replication and reverse transcription and of molecular recognition of DNA in gene regulation.

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
Hsu, Andrew; Ferrage, Fabien; Palmer 3rd, Arthur G (2018) Analysis of NMR Spin-Relaxation Data Using an Inverse Gaussian Distribution Function. Biophys J 115:2301-2309
Zeiske, Tim; Baburajendran, Nithya; Kaczynska, Anna et al. (2018) Intrinsic DNA Shape Accounts for Affinity Differences between Hox-Cofactor Binding Sites. Cell Rep 24:2221-2230
O'Brien, Paul A; Palmer 3rd, Arthur G (2018) TROSY pulse sequence for simultaneous measurement of the 15N R1 and {1H}-15N NOE in deuterated proteins. J Biomol NMR 70:205-209
Zeiske, Tim; Stafford, Kate A; Palmer 3rd, Arthur G (2016) Thermostability of Enzymes from Molecular Dynamics Simulations. J Chem Theory Comput 12:2489-92
Palmer 3rd, Arthur G (2016) A dynamic look backward and forward. J Magn Reson 266:73-80
Gill, Michelle L; Byrd, R Andrew; Palmer III, Arthur G (2016) Dynamics of GCN4 facilitate DNA interaction: a model-free analysis of an intrinsically disordered region. Phys Chem Chem Phys 18:5839-49
Palmer 3rd, Arthur G (2015) Enzyme dynamics from NMR spectroscopy. Acc Chem Res 48:457-65
Kaplan, Anna; Gaschler, Michael M; Dunn, Denise E et al. (2015) Small molecule-induced oxidation of protein disulfide isomerase is neuroprotective. Proc Natl Acad Sci U S A 112:E2245-52
Stafford, Kate A; Trbovic, Nikola; Butterwick, Joel A et al. (2015) Conformational preferences underlying reduced activity of a thermophilic ribonuclease H. J Mol Biol 427:853-66

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