Probing the dynamic process of encapsulation in Escherichia coli GroEL.

Kinetic analyses of GroE-assisted folding provide a dynamic sequence of molecular events that underlie chaperonin function. We used stopped-flow analysis of various fluorescent GroEL mutants to obtain details regarding the sequence of events that transpire immediately after ATP binding to GroEL and GroEL with prebound unfolded proteins. Characterization of GroEL CP86, a circularly permuted GroEL with the polypeptide ends relocated to the vicinity of the ATP binding site, showed that GroES binding and protection of unfolded protein from solution is achieved surprisingly early in the functional cycle, and in spite of greatly reduced apical domain movement.

Probing the functional mechanism of Escherichia coli GroEL using circular permutation.

Background: The 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 Findings: Three soluble, partially active mutants with polypeptide ends relocated into various positions of the apical domain of GroEL were isolated and studied.