Abstract
BackgroundThe Escherichia coli chaperonin GroEL subunit consists of three domains linked via two hinge regions, and each domain is responsible for a specific role in the functional mechanism. Here, we have used circular permutation to study the structural and functional characteristics of the GroEL subunit.Methodology/Principal FindingsThree soluble, partially active mutants with polypeptide ends relocated into various positions of the apical domain of GroEL were isolated and studied. The basic functional hallmarks of GroEL (ATPase and chaperoning activities) were retained in all three mutants. Certain functional characteristics, such as basal ATPase activity and ATPase inhibition by the cochaperonin GroES, differed in the mutants while at the same time, the ability to facilitate the refolding of rhodanese was roughly equal. Stopped-flow fluorescence experiments using a fluorescent variant of the circularly permuted GroEL CP376 revealed that a specific kinetic transition that reflects movements of the apical domain was missing in this mutant. This mutant also displayed several characteristics that suggested that the apical domains were behaving in an uncoordinated fashion.Conclusions/SignificanceThe loss of apical domain coordination and a concomitant decrease in functional ability highlights the importance of certain conformational signals that are relayed through domain interlinks in GroEL. We propose that circular permutation is a very versatile tool to probe chaperonin structure and function.
Highlights
Stress-induced protein denaturation and aggregation in E. coli is mitigated by the actions of various heat shock proteins, which are mobilized in response to the initial stress stimulus
We designed the CP209 mutant as an initial feasibility study by selecting a site in the GroEL subunit that would be relatively tolerant to circular permutation (i. e., not located within clearly defined alpha helices or beta sheets)
The CP254 and CP376 mutants were selected from a pool of randomly constructed candidates according to their relatively high expression in E. coli supernatant
Summary
Stress-induced protein denaturation and aggregation in E. coli is mitigated by the actions of various heat shock proteins, which are mobilized in response to the initial stress stimulus. The mechanism of protein encapsulation and release by GroEL is supported by the structure of the GroEL subunit, which consists of three distinct domains (apical, intermediate, and equatorial) that are linked by two hinge-like regions [2,3]. Recent studies have been involved in elucidating precisely how the GroEL subunit modifies its form to accomplish the complex cyclic mechanism of chaperonin action [7,8,9,10] These studies have indicated that the three domains of GroEL change their orientation relative to each other in a highly complex fashion, and that various conformational signals between domains are communicated through the two hinges that bind the domains [11,12,13]. We have used circular permutation to study the structural and functional characteristics of the GroEL subunit
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