The chaperonin GroEL in the bacterial cytoplasm has been shown to assist polypeptide chain folding but to date, a strain severely conditionally deficient in GroEL has not been available. Such a strain would allow one to address such questions as: do GroEL-deficient cells continue to translate polypeptides? How many and which polypeptides become misfolded/aggregated under such conditions? Are inclusion bodies formed? Are other chaperones induced? Here we propose to attack this problem by producing a chemical inhibitor that will cross E. coli membranes and immediately shut off the ATPase of a mutationally sensitized GroEL, blocking chaperonin action. Based on molecular modeling studies, we have selected two residues in the ATP pocket, Asn479 and Ile493, to mutate to smaller residues, alanine and glycine, to create a hydrophobic pocket potentially capable of binding one or more of a chemically synthesized series of adenine analogues with large, hydrophobic groups attached at various positions. The combination of the sensitized GroEL mutant and cell permeable inhibitor should allow for the rapid and severe inhibition of GroEL function in vivo. We will then assay protein translation, protein folding, and cell morphology of E. coli cells expressing the sensitized GroEL mutant.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Postdoctoral Individual National Research Service Award (F32)
Project #
5F32GM070139-04
Application #
7008527
Study Section
Special Emphasis Panel (ZRG1-F05 (20))
Program Officer
Portnoy, Matthew
Project Start
2004-02-01
Project End
2007-01-31
Budget Start
2006-02-01
Budget End
2007-01-31
Support Year
4
Fiscal Year
2006
Total Cost
$50,428
Indirect Cost
Name
Scripps Research Institute
Department
Type
DUNS #
781613492
City
La Jolla
State
CA
Country
United States
Zip Code
92037
Chapman, Eli; Farr, George W; Furtak, Krystyna et al. (2009) A small molecule inhibitor selective for a variant ATP-binding site of the chaperonin GroEL. Bioorg Med Chem Lett 19:811-3