Kelly Lee proposes to determine the mechanism of large scale, pH dependent reorganization of protein subunits in an RNA virus, NomegaV. Previous work in our laboratory demonstrated a remarkable size and quaternary structure change of virus-like particles made in a baculovirus expression system when the pH was lowered from 7 to 5. Kelly will use his experience in high resolution potentiometric titrations to determine the number of acidic groups being protonated during the transition. A recently prepared mutant allows the transition to be reversible and this will be studied for signs of hysteresis. Based on these and the available structure of the low pH form of the virus Kelly will propose candidates for critical acidic residues in the transition. Derek Taylor, a graduate student in the lab will make mutations to these residues and Kelly will study their phenotype. Kelly will be trained in structural methods by characterizing putative intermediates in the transition by cryoEM and image reconstruction and these will be modeled by the high resolution x-ray structure of the subunit determined in the low pH form. Kelly will also learn methods of molecular biology and baculovirus expression by making site directed mutations in the C-terminal 73 amino acid portion of the subunit, suspected of being important in the transition. This polypeptide will also be studied in solution as a fusion protein with GST to see if it has a pH dependent oligomeric behavior. Kelly has hypothesized that it is a trimer at high pH and a monomer at low pH. We anticipate that we will be able to control the transition by selected mutations and that we will map the trajectory of protein reorganization by the time Kelly has completed his fellowship.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Postdoctoral Individual National Research Service Award (F32)
Project #
5F32GM065013-02
Application #
6622380
Study Section
Biophysical Chemistry Study Section (BBCB)
Program Officer
Whitmarsh, John
Project Start
2002-02-01
Project End
Budget Start
2003-02-01
Budget End
2004-01-31
Support Year
2
Fiscal Year
2003
Total Cost
$41,608
Indirect Cost
Name
Scripps Research Institute
Department
Type
DUNS #
781613492
City
La Jolla
State
CA
Country
United States
Zip Code
92037
Dickey, Thayne H; Wuttke, Deborah S (2014) The telomeric protein Pot1 from Schizosaccharomyces pombe binds ssDNA in two modes with differing 3' end availability. Nucleic Acids Res 42:9656-65
Bloemink, Marieke; Deacon, John; Langer, Stephen et al. (2014) The hypertrophic cardiomyopathy myosin mutation R453C alters ATP binding and hydrolysis of human cardiac ?-myosin. J Biol Chem 289:5158-67
Seco, Elena M; Zinder, John C; Manhart, Carol M et al. (2013) Bacteriophage SPP1 DNA replication strategies promote viral and disable host replication in vitro. Nucleic Acids Res 41:1711-21
Dickey, Thayne H; McKercher, Marissa A; Wuttke, Deborah S (2013) Nonspecific recognition is achieved in Pot1pC through the use of multiple binding modes. Structure 21:121-132
Bloemink, Marieke J; Deacon, John C; Resnicow, Daniel I et al. (2013) The superfast human extraocular myosin is kinetically distinct from the fast skeletal IIa, IIb, and IId isoforms. J Biol Chem 288:27469-79
Dickey, Thayne H; Altschuler, Sarah E; Wuttke, Deborah S (2013) Single-stranded DNA-binding proteins: multiple domains for multiple functions. Structure 21:1074-84
Deacon, John C; Bloemink, Marieke J; Rezavandi, Heresh et al. (2012) Erratum to: Identification of functional differences between recombinant human ? and ? cardiac myosin motors. Cell Mol Life Sci 69:4239-55
Deacon, John C; Bloemink, Marieke J; Rezavandi, Heresh et al. (2012) Identification of functional differences between recombinant human ? and ? cardiac myosin motors. Cell Mol Life Sci 69:2261-77
Resnicow, Daniel I; Deacon, John C; Warrick, Hans M et al. (2010) Functional diversity among a family of human skeletal muscle myosin motors. Proc Natl Acad Sci U S A 107:1053-8
Lee, Kelly K; Gan, Lu; Tsuruta, Hiro et al. (2008) Virus capsid expansion driven by the capture of mobile surface loops. Structure 16:1491-502

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