To address the problem of protein folding, organisms have evolved molecular chaperone machines whose primary role is to foster nascent polypeptide folding by limiting aggregation. The goal of this proposal is to understand the mechanism of protein folding by the universally conserved GroEL (Hsp60) chaperone machine. Our experimental system comprises E. coli and its T4-like virulent phages. The E. coli GroEL chaperone machine is essential for bacterial viability and consists of two members, the GroEL (Hsp60) chaperone and the GroES (Hsp10) cochaperone. The T4-like phages are dependent on GroEL, but not GroES, to fold their Gp23 capsid protein, the major component of the phage head, growth-limiting and essential for phage propagation. These phages encode Gp31, a novel GroEL cochaperone that is a distant homolog of GroES and is essential for the correct folding of Gp23. The strict dependence of the phage on the host-encoded GroEL and its own Gp31 cochaperone allows us to examine GroEL/Gp31 in a manner that is not always possible in more complex systems, and is as simple as observing the formation of a plaque. Using this basic life or death genetic system, we have identified Gp39.2, a GroEL chaperone regulator also encoded by the T4-like phages. However, in contrast to Gp31, Gp39.2 is normally dispensable for laboratory T4-like phage growth, becoming absolutely essential for phage propagation only on certain groEL mutant hosts. Interestingly, Gp39.2 overexpression is bactericidal to E. coli, possibly by poisoning the GroEL machine. A series of genetic and biochemical studies are proposed to understand the unique regulation of the GroEL machine by the phage-encoded Gp31 cochaperone and Gp39.2. The genetic experiments include isolation of host and phage mutants and their compensatory mutations. The biochemical experiments include the in vitro folding of Gp23 by wild type and mutant forms of GroEL, Gp31 and Gp39.2. The use of cryo-EM imaging and X-ray crystallography of our GroEL mutants, Gp31/GroEL, GroEL/Gp39.2, and other complexes will help decipher the unique mechanisms by which Gp31 and Gp39.2 enable the GroEL chaperone to fold its Gp23 capsid substrate. Due to the extreme conservation of the Hsp60/Hsp10 machine across biological kingdoms, judged by the fact that the human machine supports E. coli growth, mechanistic insights provided by our simple E. coli/phage system will find immediate application with the Hsp60 machine of higher organisms.

Public Health Relevance

The problem of protein aggregation is universal among all living organisms. In humans it can lead to various neurodegenerative diseases, including Parkinson's and Alzheimer's. The universally conserved proteins called chaperone machines evolved to protect organisms from protein aggregation. Thus, our studies with the essential GroEL (Hsp60) chaperone machine in Escherichia coli will find immediate application to the mechanism of action of the essential human Hsp60 homolog, residing in mitochondria.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
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Prokaryotic Cell and Molecular Biology Study Section (PCMB)
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Wehrle, Janna P
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University of Utah
Schools of Medicine
Salt Lake City
United States
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Bruel, Nicolas; CastaniƩ-Cornet, Marie-Pierre; Cirinesi, Anne-Marie et al. (2012) Hsp33 controls elongation factor-Tu stability and allows Escherichia coli growth in the absence of the major DnaK and trigger factor chaperones. J Biol Chem 287:44435-46
Ang, Debbie; Georgopoulos, Costa (2012) An ORFan no more: the bacteriophage T4 39.2 gene product, NwgI, modulates GroEL chaperone function. Genetics 190:989-1000
Perrody, Elsa; Cirinesi, Anne-Marie; Desplats, Carine et al. (2012) A bacteriophage-encoded J-domain protein interacts with the DnaK/Hsp70 chaperone and stabilizes the heat-shock factor ?32 of Escherichia coli. PLoS Genet 8:e1003037