My proposed research will focus on the investigation of communication between NG and M domains of E. co/i Ffh protein upon signal sequence recognition and binding. Proteins which are synthesized within the cell must be correctly targeted for proper localization. Prokaryotic cell signal recognition particle (SRP) is composed of a protein component termed Ffh and 4.5S RNA. AU SRP component are essential for cell growth. The initial step in the SRP-mediated secretory pathway involves recognition of the signal sequence. Ffh possesses GTPase activity localized in the NG domain, which is known to bind subsequently to its membrane receptor FtsY. Detailed mechanisms of this cascade of interactions are still largely unknown. I will attempt to address only one aspect within these multi-component interactions. Namely, the emerging possibility of a structural rearrangement of the NG and M domains upon signal sequence binding will be addressed.
This aim will be accomplished using a combination of mutagenesis of Fth for labeling purposes, steady-state and time-resolved fluorescence spectroscopy to assess the change in the interdomain distances upon signal peptide binding, fluorescence quenching and anisotropy measurements to determine the accessibility and mobility of the chromophores. The present system is chosen for its relative simplicity in handling and for its high homology to the eukaryotic SRP, thus results can be extended to the mammalian SRP machinery.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Postdoctoral Individual National Research Service Award (F32)
Project #
5F32GM066591-02
Application #
6612526
Study Section
Special Emphasis Panel (ZRG1-F04 (20))
Program Officer
Whitmarsh, John
Project Start
2002-07-10
Project End
2004-07-09
Budget Start
2003-07-10
Budget End
2004-07-09
Support Year
2
Fiscal Year
2003
Total Cost
$46,420
Indirect Cost
Name
University of Massachusetts Amherst
Department
Biochemistry
Type
Schools of Arts and Sciences
DUNS #
153926712
City
Amherst
State
MA
Country
United States
Zip Code
01003