CIpXP and other AAA+ proteases recognize, mechanically unfold, and translocate target proteins into a chamber for proteolysis. It is not known if these remarkable molecular machines operate by a stochastic or sequential mechanism or how power strokes relate to the ATP-hydrolysis cycle. Single-molecule optical trapping allows CIpXP unfolding to be directly visualized and reveals translocation steps of ~1–4 nm in length, but how these activities relate to solution degradation and the physical properties of substrate proteins remains unclear. By studying single-molecule degradation using different multi-domain substrates and CIpXP variants, we answer many of these questions and provide evidence for stochastic unfolding and translocation. We also present a mechanochemical model that accounts for single-molecule, biochemical, and structural results, for our observation of enzymatic memory in translocation stepping, for the kinetics of translocation steps of different sizes, and for probabilistic but highly coordinated subunit activity within the CIpX ring.

Many biological active proteins are assembled in protein complexes. Understanding the (dis)assembly of such complexes is therefore of major interest. Here we use mass spectrometry to monitor the disassembly induced by thermal activation of the heptameric co-chaperonins GroES and gp31. We use native electrospray ionization mass spectrometry (ESI-MS) on a Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometer to monitor the stoichiometry of the chaperonins. A thermally controlled electrospray setup was employed to analyze conformational and stoichiometric changes of the chaperonins at varying temperature. The native ESI-MS data agreed well with data obtained from fluorescence spectroscopy as the measured thermal dissociation temperatures of the complexes were in good agreement. Furthermore, we observed that thermal denaturing of GroES and gp31 proceeds via intermediate steps of all oligomeric forms, with no evidence of a transiently stable unfolded heptamer. We also evaluated the thermal dissociation of the chaperonins in the gas phase using infrared multiphoton dissociation (IRMPD) for thermal activation. Using gas-phase activation the smaller (2-4) oligomers were not detected, only down to the pentamer, whereafter the complex seemed to dissociate completely. These results demonstrate clearly that conformational changes of GroES and gp31 due to heating in solution and in the gas phase are significantly different.

The biological motor ClpXP pulls against a targeted protein until it mechanically unfolds and translocates the polypeptide into a chamber for degradation. We directly monitor degradation of model polyprotein substrates in dual-trap single molecule assays.

Propagation of bacteriophage T4 in its host Escherichia coli involves the folding of the major capsid protein gp23, which is facilitated by a hybrid chaperone complex consisting of the bacterial chaperonin GroEL and the phage-encoded co-chaperonin, gp31. It has been well established that the GroEL-gp31 complex is capable of folding gp23 whereas the homologous GroEL-GroES complex cannot perform this function. To assess whether this is a consequence of differences in the interactions of the proteins within the chaperonin complex, we have investigated the dissociation kinetics of GroEL-gp31 and GroEL-GroES complexes using Forster resonance energy transfer. Here we report that the dissociation of gp31 from GroEL is slightly faster than that of GroES from GroEL and is further accelerated by the binding of gp23. In contrast to what had been observed previously, we found that gp23 is able to interact with the GroEL-GroES complex, which might explain how bacteriophage T4 redirects the folding machinery of Escherichia coli during morphogenesis.