Single-molecule study of chaperonin cooperativity with anti-brownian electrokinetic trap [electronic resource]
Keywords: Applied Physics

Group II chaperonins are cellular protein-folding nanomachines with a stacked double-ring structure and two built-in lids. Each of the rings consists of eight or nine subunits, defining a cavity that can be closed with the lid. Chaperonins adopt distinct open and closed structures. The open structure allows substrates to bind, the closed structure encloses and folds the substrates, and ATP-regulated conformational changes between the open and closed structures are critical for the timing of the enzymatic cycle. These conformational changes are collective movements of multiple subunits and therefore highly depend on the cooperativity between the subunits. Although ensemble-averaged ATPase rates have indicated the existence of positive and negative cooperativities, the molecular mechanisms remain unknown. The present study provides more detailed understanding of the cooperativities in two group II chaperonins, TRiC and MmCpn, by measuring the stoichiometry of the various forms of nucleotides at the single-molecule level. To measure the nucleotide stoichiometry, each ATP was labeled with a Cy3 fluorescent dye and the Cy3-nucleotide-bound chaperonins were trapped in an Anti-Brownian ELectrokinetic trap (ABEL trap) for detailed study. The ABEL trap can localize a single fluorescently labeled protein for seconds in free solution, allowing single-molecule study of chaperonins without immobilization-induced artifacts. Trapped chaperonins containing multiple Cy3-nucleotides produce step-wise-decreasing intensity traces corresponding to the photobleaching of individual dyes, allowing extraction of nucleotide number distributions. The distributions show that the eukaryotic chaperonin TRiC/CCT, consisting of two copies of eight different subunits, hydrolyzes either zero or eight ATPs over a wide range of ATP concentrations. Although the ensemble-averaged results agree with standard cooperative ATP binding models, the single-molecule distributions do not: at low ATP concentrations, the preference for each chaperonin to hydrolyze eight ATPs is too strong to be explained by ATP binding alone and requires modeling the cooperativity in hydrolysis as well. In addition, the time-dependent ADP number distribution suggests simultaneous release of all eight ADPs governed by a one-step reaction that is the rate-limiting step of the ATPase cycle. Since TRiC is a hetero-oligomer, the fact that only half of the subunits hydrolyze ATP can be a consequence of the heterogeneous ATP binding affinities, the allosteric regulation or a combination of both. But strikingly, MmCpn, a homo-16-mer archaeal chaperonin, also hydrolyzes about eight ATPs at saturating ATP concentrations, suggesting that allosteric regulation heavily contributes to the determination of hydrolysis-active subunits. However, unlike TRiC, the number of ATPs hydrolyzed by each MmCpn quickly decreases when the ATP concentration decreases, consistent with a standard cooperative binding model. In addition, these distributions become less stable when the sample incubation time is shortened, suggesting sequential hydrolysis of the bound ATPs and temporary closure of the chaperonin containing a mixture of hydrolyzed and unhydrolyzed ATPs. This method of counting fluorescent dyes on ABEL-trapped proteins can be also extended to measure the nucleotide or substrate stoichiometry in other multi-subunit enzymes. With the native activities maximally preserved, the obtained single-molecule information will greatly improve our understanding of enzymatic mechanisms.