Abstract
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. Analysis of fluorescent GroEL SR-1 and GroEL D398A variants suggested that among other factors, the presence of two GroEL rings and a specific conformational rearrangement of Helix M in GroEL contribute significantly to the rapid release of unfolded protein from the GroEL apical domain.
Highlights
Analyses of macromolecular complexes such as the E. coli chaperonin GroE involve a multitude of functionally relevant events, dynamic transitions of the molecular machinery, and various structural characteristics of the molecular architecture that must all be united coherently and consistently in order to understand the underlying molecular mechanism in detail
We show results obtained from fluorescent variants of the ATPase-impaired mutant D398A (Fig. 1, center right) [10,11,12] and the single-ring GroEL mutant, SR-1 (Fig. 1, bottom right) [8,9] that suggest that encapsulation of unfolded proteins by GroE is essentially a multi-stage sequential process whose smooth execution is dependent on numerous structural factors of the GroEL architecture
Application of random circular permutation to the E. coli GroEL subunit [22] produced a mutant (GroEL CP376) that was used in a previous study to demonstrate that Phase C exclusively reflects localized movements of the GroEL apical domain
Summary
Analyses of macromolecular complexes such as the E. coli chaperonin GroE involve a multitude of functionally relevant events, dynamic transitions of the molecular machinery, and various structural characteristics of the molecular architecture that must all be united coherently and consistently in order to understand the underlying molecular mechanism in detail. Many studies have succeeded in elucidating and correlating these varied details of the chaperonin to form a well defined overview of the molecular mechanism, and recent experiments are involved in forming increasingly detailed views of various segments of the mechanism [1,2,3]. One such segment involves the process by which GroEL transfers proteins into the protective central cavity formed by the unique double ringed quaternary structure of the 14 GroEL subunits [4,5,6,7]. From a principle of minimum structural alterations between substates, they postulated a sequential mechanism that describes the initial molecular trajectory of GroE encapsulation that culminates in GroES binding and formation of the capsule
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