My goal is a quantitative understanding of the activation of Arp2/3 complex, a protein assembly instrumental in cellular motility. I propose three lines of experiments to understand how activators change the conformation of Arp2/3 complex and how this conformational change influences actin filament nucleation and branching in vivo and in vitro. . Use disulfide crosslinking between cysteine residues engineered into fission yeast Arp2/3 complex to create constitutively active and inactive Arp2/3 complex. I will compare native Arp2/3 complex and these two frozen conformations in spectroscopic and single particle fluorescence microscopy assays to analyze the kinetics and thermodynamics of Arp2/3 complex activation by nucleation promoting factors such as the VGA regions of WASp and Scar, actin monomers, and pre-existing actin filaments. . Use high-resolution 3D fluorescence microscopy to characterize the effects of a panel of mutations on the ability of Arp2/3 complex to support actin filament nucleation in live fission yeast. Use simulations to study Arp2/3 subunit mechanisms involved in nucleotide binding and activation.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Postdoctoral Individual National Research Service Award (F32)
Project #
5F32GM074504-03
Application #
7229011
Study Section
Special Emphasis Panel (ZRG1-F05 (20))
Program Officer
Rodewald, Richard D
Project Start
2005-05-01
Project End
2008-04-30
Budget Start
2007-05-01
Budget End
2008-04-30
Support Year
3
Fiscal Year
2007
Total Cost
$50,428
Indirect Cost
Name
Yale University
Department
Biochemistry
Type
Schools of Arts and Sciences
DUNS #
043207562
City
New Haven
State
CT
Country
United States
Zip Code
06520
Dalhaimer, Paul; Pollard, Thomas D (2010) Molecular dynamics simulations of Arp2/3 complex activation. Biophys J 99:2568-76
Dalhaimer, Paul; Pollard, Thomas D; Nolen, Brad J (2008) Nucleotide-mediated conformational changes of monomeric actin and Arp3 studied by molecular dynamics simulations. J Mol Biol 376:166-83