Single-Molecule Studies of Chaperonin Mechanism

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DESCRIPTION (provided by applicant): The goal of this exploratory R21 proposal is to develop and use single-molecule approaches to understand the molecular mechanisms of chaperonin function. We will focus on two cooperative oligomeric enzymes, GroEL/ES and TRiC, both of which are essential to the intracellular folding of a variety of newly-translated proteins and enzymes. By comparing bulk measurements with the dynamical sequencing behavior of single molecules, we will obtain insight into the timing and dynamics of the conformational changes that drive the function of these enzymes. Double-ring chaperonins bind unfolded polypeptides within their ring cavities, prevent off-pathway reactions, and promote productive protein folding to the native state in a highly cooperative, ATP-dependent manner. The homo-oligomeric E. coli complex GroEL acts in concert with the single-ring complex GroES. Unlike GroEL, the eukaryotic cytosolic chaperonin, denoted CCT or TRiC, is hetero-oligomeric and functions without a GroES-like cofactor. The primary objective of the proposed work is to explore the specifics of dynamical sequencing of the conformational states of these enzymes. This goal will be addressed by applying both biological and physical expertise of the researchers to two specific aims focused on understanding aspects of chaperonin action. By selecting individual copies of chaperonin complexes, we will remove heterogeneity between individual copies that are in different conformational states. In this way we will be able to sense and characterize the sequence of dynamical events and local environmental changes that occur during the nucleotide cycle, as chaperones assist in the folding of selected substrate proteins. Aim 1 will investigate hypotheses relating to the mechanism of action of assisted protein folding in the bacterial GroEL/GroES chaperonin system. Aim 2 will address similar questions for the less-studied but equally important eukaryotic chaperonin, TriC. The exploratory nature of this application arises from the fact that many of the assays proposed have not been previously utilized to explore chaperonin mechanisms.