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 184.108.40.206, 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.
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.
|Ergel, Burçe; Gill, Michelle L; Brown, Lewis et al. (2014) Protein dynamics control the progression and efficiency of the catalytic reaction cycle of the Escherichia coli DNA-repair enzyme AlkB. J Biol Chem 289:29584-601|
|Stafford, Kate A; Robustelli, Paul; Palmer 3rd, Arthur G (2013) Thermal adaptation of conformational dynamics in ribonuclease H. PLoS Comput Biol 9:e1003218|
|Zeiske, Tim; Stafford, Kate A; Friesner, Richard A et al. (2013) Starting-structure dependence of nanosecond timescale intersubstate transitions and reproducibility of MD-derived order parameters. Proteins 81:499-509|
|Robustelli, Paul; Stafford, Kate A; Palmer 3rd, Arthur G (2012) Interpreting protein structural dynamics from NMR chemical shifts. J Am Chem Soc 134:6365-74|
|Gill, Michelle L; Palmer 3rd, Arthur G (2011) Multiplet-filtered and gradient-selected zero-quantum TROSY experiments for 13C1H3 methyl groups in proteins. J Biomol NMR 51:245-51|
|Trbovic, Nikola; Cho, Jae-Hyun; Abel, Robert et al. (2009) Protein side-chain dynamics and residual conformational entropy. J Am Chem Soc 131:615-22|
|Trbovic, Nikola; Kim, Byungchan; Friesner, Richard A et al. (2008) Structural analysis of protein dynamics by MD simulations and NMR spin-relaxation. Proteins 71:684-94|
|Koehnke, Jesko; Jin, Xiangshu; Trbovic, Nikola et al. (2008) Crystal structures of beta-neurexin 1 and beta-neurexin 2 ectodomains and dynamics of splice insertion sequence 4. Structure 16:410-21|
|Maragakis, Paul; Lindorff-Larsen, Kresten; Eastwood, Michael P et al. (2008) Microsecond molecular dynamics simulation shows effect of slow loop dynamics on backbone amide order parameters of proteins. J Phys Chem B 112:6155-8|
|Trott, Oleg; Siggers, Keri; Rost, Burkhard et al. (2008) Protein conformational flexibility prediction using machine learning. J Magn Reson 192:37-47|
Showing the most recent 10 out of 30 publications