The long-term objective of this research program is to understand the structural and dynamic bases of protein recognition leading to the assembly of viruses. This knowledge will permit identification of steps along the assembly pathways that can be targeted for interference and control. The plan utilizes static and dynamic Raman spectroscopic probes in combination with complementary hydrodynamic and calorimetric methods to elucidate protein structures and interactions.
The specific aims of the proposed research are as follows: (1) Identify protein main-chain conformations, side-chain interactions and covalent modifications that regulate the assembly and function of the specialized portal vertices of icosahedral viruses. (2) Determine new structural information on the viral terminase complexes that function as ATP-fueled motors to package viral genomes within capsids. (3) Ascertain kinetic and thermodynamic controls of discrete steps in viral capsid morphogenesis, especially those relating to procapsid formation, capsid maturation and stabilization of the capsid/genome interface. (4) Establish new qualitative and quantitative correlations to advance the effectiveness of Raman spectroscopy as a diagnostic probe of viral protein structures and assembly pathways. The investigations will focus on key viruses for which complementary genetic and biochemical data are now available or obtainable, including isometric dsDNA and dsRNA virions (P22, HK97, PRD1, 06), filamentous virions (fd,Pf1,Pf3, PH75) and related subviral assemblies. The project will exploit novel probes of protein structure, dynamics and interactions within the framework of the virus assembly/disassembly paradigm. Experimental protocols will incorporate isotope-edited Raman (IER), near-infrared (NIR)laser Raman, ultraviolet-resonance Raman (UVRR) and polarized Raman spectroscopies, including temperature- dependent and H/D exchange-dependent applications. The plan is designed specifically to advance understanding of the assembly roles of viral subunit folding, structure, dynamics and recognition, and generally to expand the capabilities of laser Raman spectroscopy as a probe of biological complexes. The biological significance of the proposed research derives from the critical need for fundamental knowledge about the molecular basis of virus assembly in order to understand and control viral diseases.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM050776-34
Application #
7493074
Study Section
Macromolecular Structure and Function C Study Section (MSFC)
Program Officer
Basavappa, Ravi
Project Start
1987-09-01
Project End
2010-08-31
Budget Start
2008-09-01
Budget End
2010-08-31
Support Year
34
Fiscal Year
2008
Total Cost
$295,284
Indirect Cost
Name
University of Missouri Kansas City
Department
Anatomy/Cell Biology
Type
Schools of Arts and Sciences
DUNS #
010989619
City
Kansas City
State
MO
Country
United States
Zip Code
64110
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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