DEVELOPING A NEW MODEL OF THE GROEL FUNCTIONAL CYCLE AND ITS IMPLICATIONS FOR THE GROEL-OPTIMIZED SUBSTRATE PROTEIN REFOLDING

Abstract

Despite years of research work, many aspects of the fundamentally important GroEL functional cycle are still in dispute. The work of this dissertation mainly focuses on three major disputes in the field: the identity of the rate determining step (RDS), the physiological order of arrival of ligands (ATP, SP and GroES) to the GroEL trans ring, and the role of the symmetric GroEL-GroES2 "football" complex in the overall chaperonin cycle. With multiple carefully designed spectroscopic probes, a pre-steady state survey has been conducted on the kinetics of the GroEL functional cycle. From the survey, a two cycle model emerges: in the absence of SP, ADP release is the RDS of the asymmetric cycle and consequently, the asymmetric GroEL-GroES1 ,"bullet" which precedes this step, is the pre-dominant species. In this mode, the machine turns over very slowly, minimizing futile ATP consumption. Due to the slow release of ADP, the system turns over in a well defined manner with the two rings operating 180o out of phase of each other, analogous to a two-stroke motor. In the symmetric cycle, which operates in the presence of SP, the release of ADP is greatly accelerated while the intrinsic ATPase activity of GroEL remains unaffected. Consequently ATP hydrolysis becomes the RDS and the symmetric GroEL-GroES2 "football" becomes the predominant species. Contrary to previous chaperonin dogma, the symmetric complex is a highly dynamic species exchanging its two bound ligands, GroES and encapsulated SP from both rings with a half time ~1sec. Switching to a parallel processing machine, the chaperonins turns over rapidly, ultimately driven by stochastic hydrolysis of ATP which causes the symmetric complex to undergo breakage of symmetry (BoS). With such a dynamic system, folding in the `folding cage' seems less important in GroEL-mediated SP refolding as suggested by the passive refolding model. Instead, GroEL may play a more active role in achieving its central biological function as indicated by this two cycle model. This may be the very reason why employing even as low as one GroEL ring per ten SP can achieve SP refolding to a similar extent as using a stoichiometric amount.