The central objective of the proposed research is to determine the molecular basis for protein-nucleic acid recognition which leads to the proper assembly and stability of viruses and gene regulatory complexes.
The specific aims are to determine, for native viruses and macromolecular assemblies, the following: (i) specific molecular subgroups which are responsible for protein-nucleic acid recognition, (ii) chemical and physical factors, such as pH, ionic composition, and temperature, which regulate assembly and disassembly processes, and (iii) secondary structures and conformations of nucleic acid and protein molecules at physiological conditions.
These aims shall be pursued primarily by the method of laser Raman spectroscopy, employing the powerful technique of optical multichannel analysis (OMA). Targeted for study are a number of viruses and nucleoproteins for which complementary genetic, biochemical and structural information is available or obtainable. Among these systems are filamentious bacteriophages, isometric viruses, and phage lambda cI and Cro repressors and their recognition operators. The interpretation of results on viruses and nucleoproteins will be aided by parallel studies of model DNA and protein molecules. The collection of Raman spectroscopic data by the OMA rapid acquisition technique will enable the investigation of both equilibrium structures and dynamic processes. Structures of viruses and nucleoproteins will be determined and compared in both the solution and crystal states. The approach is one which can test the applicability of X-ray determined crystal structures for aqueous nucleic acids, proteins and their biologically active assemblies. Since many of the results will be applicable to viruses which infect higher organisms, this research program has long range 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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