The central objective of the proposed research is to understand the molecular basis of protein and nucleic acid recognition leading to the proper assembly and stability of viruses. This knowledge will help to identify steps in viral assembly mechanisms that may be vulnerable to interference and control.
The specific aims are to: 1. Identify protein conformations and residues which regulate subunit assembly and disassembly, stabilize virions and precursor structures, and mediate genome packaging at physiological conditions. 2. Identify molecular subgroups which can be altered along the viral assembly pathway by controlled changes in cellular and/or physiological factors, such as pH, ionic composition and temperature. 3. Determine the dynamics of genome packaging reactions and the kinetics of hydrogen-isotope exchange reactions in viruses, viral precursors and their components as specific probes of assembly. 4. Establish new qualitative and quantitative correlations between the data of Raman spectroscopy and the structures and interactions of proteins in viruses and in other macromolecular assemblies These aims will be pursued using recently developed Raman and ultraviolet-resonance Raman (UVRR) spectroscopic methods, including (a) a Raman microscope for determining residue orientations in viral assemblies, (b) a Raman microdialysis flow cell for determining the kinetics of H-D exchange reactions in viral constituents and for time-resolving specific steps along viral assembly pathways, and (c) a UVRR spectrometer custom-designed for novel structural investigations of viruses and their components. Targeted for study are key virions for which complementary genetic, biochemical and structural information is available or obtainable, including filamentous (fd, Pf1, Pf3) and isometric viruses (HSV-1, P22, PRD1, phi6, phi29). The biological significance of this research derives from the need for fundamental information about the role of molecular recognition in mechanisms of viral assembly. Since he knowledge gained will be applicable to viruses that infect higher organisms, including humans, this research has long-term health-related benefits.
|Nemecek, Daniel; Stepanek, Josef; Thomas Jr, George J (2013) Raman spectroscopy of proteins and nucleoproteins. Curr Protoc Protein Sci Chapter 17:Unit17.8|
|Tsuboi, Masamichi; Tsunoda, Masaru; Overman, Stacy A et al. (2010) A structural model for the single-stranded DNA genome of filamentous bacteriophage Pf1. Biochemistry 49:1737-43|
|Nemecek, Daniel; Overman, Stacy A; Hendrix, Roger W et al. (2009) Unfolding thermodynamics of the Delta-domain in the prohead I subunit of phage HK97: determination by factor analysis of Raman spectra. J Mol Biol 385:628-41|
|Tsuboi, Masamichi; Benevides, James M; Thomas Jr, George J (2009) Raman tensors and their application in structural studies of biological systems. Proc Jpn Acad Ser B Phys Biol Sci 85:83-97|
|Nemecek, Daniel; Lander, Gabriel C; Johnson, John E et al. (2008) Assembly architecture and DNA binding of the bacteriophage P22 terminase small subunit. J Mol Biol 383:494-501|
|Nemecek, Daniel; Gilcrease, Eddie B; Kang, Sebyung et al. (2007) Subunit conformations and assembly states of a DNA-translocating motor: the terminase of bacteriophage P22. J Mol Biol 374:817-36|
|Hammel, Michal; Nemecek, Daniel; Keightley, J Andrew et al. (2007) The Staphylococcus aureus extracellular adherence protein (Eap) adopts an elongated but structured conformation in solution. Protein Sci 16:2605-17|
|Sun, Ying; Overman, Stacy A; Thomas Jr, George J (2007) Impact of in vitro assembly defects on in vivo function of the phage P22 portal. Virology 365:336-45|
|Wang, Ying A; Yu, Xiong; Overman, Stacy et al. (2006) The structure of a filamentous bacteriophage. J Mol Biol 361:209-15|
|Tsuboi, Masamichi; Benevides, James M; Bondre, Priya et al. (2005) Structural details of the thermophilic filamentous bacteriophage PH75 determined by polarized Raman microspectroscopy. Biochemistry 44:4861-9|
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