Ribosome recycling is the final step of the cyclic process of translation, where the post-termination complex (PoTC) is disassembled by the concerted action of ribosome recycling factor (RRF) and elongation factor G (EF-G) in the sub-second time range. Since, however, both the RRF and PoTC display highly dynamic action during this process, it is difficult to assess the molecular details of the interactions between the factors and the ribosome that are essential for rapid subunit separation. Here we characterized the molecular dynamics of RRF and PoTC by combined use of molecular dynamics simulations, single molecule fluorescence detection and single-particle cryo-EM analysis, with time resolutions in the sub-millisecond to minute range. We found that RRF displays two-layer dynamics: intra- and inter-molecular dynamics during ribosome splitting. The intra-molecular dynamics exhibits two different configurations of RRF: \'bent\' and \'extended\'. A single-site mutant of RRF increases its propensity to the \'extended\' conformation and leads to a higher binding affinity of RRF to the PoTC. The inter-molecular dynamics between RRF and EF-G in the PoTC reveals that the domain IV of EF-G pushes against the domain II of RRF, triggering the disruption of the major inter-subunit bridge B2a, and catalyzes the splitting.

Hsp70 is a conserved molecular chaperone that plays an indispensable role in regulating protein folding, translocation, and degradation. The conformational dynamics of Hsp70 and its regulation by cochaperones are vital to its function. Using bulk and single-molecule fluorescence resonance energy transfer (smFRET) techniques, we studied the interdomain conformational distribution of human stress-inducible Hsp70A1 and the kinetics of conformational changes induced by nucleotide and the Hsp40 cochaperone Hdj1. We found that the conformations between and within the nucleotide- and substrate-binding domains show heterogeneity. The conformational distribution in the ATP-bound state can be induced by Hdj1 to form an \"ADP-like\" undocked conformation, which is an ATPase-stimulated state. Kinetic measurements indicate that Hdj1 binds to monomeric Hsp70 as the first step, then induces undocking of the two domains and closing of the substrate-binding cleft. Dimeric Hdj1 then facilitates dimerization of Hsp70 and formation of a heterotetrameric Hsp70-Hsp40 complex. Our results provide a kinetic view of the conformational cycle of Hsp70 and reveal the importance of the dynamic nature of Hsp70 for its function.

The phosphoenolpyruvate-dependent phosphotransferase system (PTS) transports sugar into bacteria and phosphorylates the sugar for metabolic consumption. The PTS is important for the survival of bacteria and thus a potential target for antibiotics, but its mechanism of sugar uptake and phosphorylation remains unclear. The PTS is composed of multiple proteins, and the membrane-embedded Enzyme IIC (EIIC) component transports sugars across the membrane. Crystal structures of two members of the glucose superfamily of EIICs, bcChbC and bcMalT, were solved in the inward-facing and outward-facing conformations, and the structures suggest that sugar translocation could be achieved by movement of a structured domain that contains the sugar-binding site. However, different conformations have not been captured on the same transporter to allow precise description of the conformational changes. Here we present a crystal structure of bcMalT trapped in an inward-facing conformation by a mercury ion that bridges two strategically placed cysteine residues. The structure allows direct comparison of the outward- and inward-facing conformations and reveals a large rigid-body motion of the sugar-binding domain and other conformational changes that accompany the rigid-body motion. All-atom molecular dynamics simulations show that the inward-facing structure is stable with or without the cross-linking. The conformational changes were further validated by single-molecule Föster resonance energy transfer (smFRET). Combined, these results establish the elevator-type mechanism of transport in the glucose superfamily of EIIC transporters.

Determining the energetics of the unfolded state of a protein is essential for understanding the folding mechanics of ordered proteins and the structure-function relation of intrinsically disordered proteins. Here, we adopt a coil-globule transition theory to develop a general scheme to extract interaction and free energy information from single-molecule fluorescence resonance energy transfer spectroscopy. By combining protein stability data, we have determined the free energy difference between the native state and the maximally collapsed denatured state in a number of systems, providing insight on the specific/nonspecific interactions in protein folding. Both the transfer and binding models of the denaturant effects are demonstrated to account for the revealed linear dependence of inter-residue interactions on the denaturant concentration, and are thus compatible under the coil-globule transition theory to further determine the dimension and free energy of the conformational ensemble of the unfolded state. The scaling behaviors and the effective θ-state are also discussed.