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Many essential proteins cannot fold without help from chaperonins, like the GroELS system of Escherichia coli . How chaperonins accelerate protein folding remains controversial. Here we test key predictions of both passive and active models of GroELS-stimulated folding, using the endogenous E. coli metalloprotease PepQ. While GroELS increases the folding rate of PepQ by over 15-fold, we demonstrate that slow spontaneous folding of PepQ is not caused by aggregation. Fluorescence measurements suggest that, when folding inside the GroEL-GroES cavity, PepQ populates conformations not observed during spontaneous folding in free solution. Using cryo-electron microscopy, we show that the GroEL C-termini make physical contact with the PepQ folding intermediate and help retain it deep within the GroEL cavity, resulting in reduced compactness of the PepQ monomer. Our findings strongly support an active model of chaperonin-mediated protein folding, where partial unfolding of misfolded intermediates plays a key role. In the prevailing model for assisted protein folding, chaperonins act passively by preventing protein aggregation. Here, the authors use single-molecule fluorescence measurements and cryo-electron microscopy and show that the E. coli GroELS chaperonin system also has an active role in folding the endogenous bacterial protein PepQ.

Prolidases, metalloproteases that catalyze the cleavage of Xaa-Pro dipeptides, are conserved enzymes found in prokaryotes and eukaryotes. In humans, prolidase is crucial for the recycling of collagen. To further characterize the essential elements of this enzyme, we utilized the Escherichia coli prolidase, PepQ, which shares striking similarity with eukaryotic prolidases. Through structural and bioinformatic insights, we have extended previous characterizations of the prolidase active site, uncovering a key component for substrate specificity. Here we report the structure of E. coli PepQ, solved at 2.0 Å resolution. The structure shows an antiparallel, dimeric protein, with each subunit containing N-terminal and C-terminal domains. The C-terminal domain is formed by the pita-bread fold typical for this family of metalloproteases, with two Mg(II) ions coordinated by five amino-acid ligands. Comparison of the E. coli PepQ structure and sequence with homologous structures and sequences from a diversity of organisms reveals distinctions between prolidases from Gram-positive eubacteria and archaea, and those from Gram-negative eubacteria, including the presence of loop regions in the E. coli protein that are conserved in eukaryotes. One such loop contains a completely conserved arginine near the catalytic site. This conserved arginine is predicted by docking simulations to interact with the C-terminus of the substrate dipeptide. Kinetic analysis using both a charge-neutralized substrate and a charge-reversed variant of PepQ support this conclusion, and allow for the designation of a new role for this key region of the enzyme active site.

Vital cellular processes, from cell growth to synaptic transmission, rely on membrane-bounded carriers and vesicles to transport molecular cargo to and from specific intracellular compartments throughout the cell. Compartment-specific proteins are required for the final step, membrane fission, which releases the transport carrier from the intracellular compartment. The role of fission proteins, especially at intracellular locations and in non-neuronal cells, while informed by the dynamin-1 paradigm, remains to be resolved. In this study, we introduce a highly sensitive approach for the identification and analysis of membrane fission machinery, called burst analysis spectroscopy (BAS). BAS is a single particle, free-solution approach, well suited for quantitative measurements of membrane dynamics. Here, we use BAS to analyze membrane fission induced by the potent, fission-active ENTH domain of epsin. Using this method, we obtained temperature-dependent, time-resolved measurements of liposome size and concentration changes, even at sub-micromolar concentration of the epsin ENTH domain. We also uncovered, at 37°C, fission activity for the full-length epsin protein, supporting the argument that the membrane-fission activity observed with the ENTH domain represents a native function of the full-length epsin protein.

Many proteins cannot fold without the assistance of chaperonin machines like GroEL and GroES. The nature of this assistance, however, remains poorly understood. Here we demonstrate that unfolding of a substrate protein by GroEL enhances protein folding. We first show that capture of a protein on the open ring of a GroEL–ADP–GroES complex, GroEL's physiological acceptor state for non-native proteins in vivo , leaves the substrate protein in an unexpectedly compact state. Subsequent binding of ATP to the same GroEL ring causes rapid, forced unfolding of the substrate protein. Notably, the fraction of the substrate protein that commits to the native state following GroES binding and protein release into the GroEL–GroES cavity is proportional to the extent of substrate-protein unfolding. Forced protein unfolding is thus a central component of the multilayered stimulatory mechanism used by GroEL to drive protein folding.

Recent structural and biochemical investigations have come together to allow a better understanding of the mechanism of chaperonin (GroEL, Hsp60)-mediated protein folding, the final step in the accurate expression of genetic information. Major, asymmetric conformational changes in the GroEL double toroid accompany binding of ATP and the cochaperonin GroES. When a nonnative polypeptide, bound to one of the GroEL rings, is encapsulated by GroES to form a cis ternary complex, these changes drive the polypeptide into the sequestered cavity and initiate its folding. ATP hydrolysis in the cis ring primes release of the products, and ATP binding in the trans ring then disrupts the cis complex. This process allows the polypeptide to achieve its final native state, if folding was completed, or to recycle to another chaperonin molecule, if the folding process did not result in a form committed to the native state.

The chaperonin GroEL is a double-ring structure with a central cavity in each ring that provides an environment for the efficient folding of proteins when capped by the co-chaperone GroES in the presence of adenine nucleotides. Productive folding of the substrate rhodanese has been observed in cis ternary complexes, where GroES and polypeptide are bound to the same ring, formed with either ATP, ADP or non-hydrolysable ATP analogues, suggesting that the specific requirement for ATP is confined to an action in the trans ring that evicts GroES and polypeptide from the cis side. We show here, however, that for the folding of malate dehydrogenase and Rubisco there is also an absolute requirement for ATP in the cis ring, as ADP and AMP-PNP are unable to promote folding. We investigated the specific roles of binding and hydrolysis of ATP in the cis and trans rings using mutant forms of GroEL that bind ATP but are defective in its hydrolysis. Binding of ATP and GroES in cis initiated productive folding inside a highly stable GroEL-ATP-GroES complex. To discharge GroES and polypeptide, ATP hydrolysis in the cis ring was required to form a GroEL-ADP-GroES complex with decreased stability, priming the cis complex for release by ATP binding (without hydrolysis) in the trans ring. These observations offer an explanation of why GroEL functions as a double-ring complex.

Protein folding is a spontaneous process that is essential for life, yet the concentrated and complex interior of a cell is an inherently hostile environment for the efficient folding of many proteins. Some proteins-constrained by sequence, topology, size, and function-simply cannot fold by themselves and are instead prone to misfolding and aggregation. This problem is so deeply entrenched that a specialized family of proteins, known as molecular chaperones, evolved to assist in protein folding. Here we examine one essential class of molecular chaperones, the large, oligomeric, and energy utilizing chaperonins or Hsp60s. The bacterial chaperonin GroEL, along with its co-chaperonin GroES, is probably the best-studied example of this family of protein-folding machine. In this review, we examine some of the general properties of proteins that do not fold well in the absence of GroEL and then consider how folding of these proteins is enhanced by GroEL and GroES. Recent experimental and theoretical studies suggest that chaperonins like GroEL and GroES employ a combination of protein isolation, unfolding, and conformational restriction to drive protein folding under conditions where it is otherwise not possible.

Productive cis folding by the chaperonin GroEL is triggered by the binding of ATP but not ADP, along with cochaperonin GroES, to the same ring as non‐native polypeptide, ejecting polypeptide into an encapsulated hydrophilic chamber. We examined the specific contribution of the γ‐phosphate of ATP to this activation process using complexes of ADP and aluminium or beryllium fluoride. These ATP analogues supported productive cis folding of the substrate protein, rhodanese, even when added to already‐formed, folding‐inactive cis ADP ternary complexes, essentially introducing the γ‐phosphate of ATP in an independent step. Aluminium fluoride was observed to stabilize the association of GroES with GroEL, with a substantial release of free energy (−46 kcal/mol). To understand the basis of such activation and stabilization, a crystal structure of GroEL–GroES–ADP·AlF 3 was determined at 2.8 Å. A trigonal AlF 3 metal complex was observed in the γ‐phosphate position of the nucleotide pocket of the cis ring. Surprisingly, when this structure was compared with that of the previously determined GroEL–GroES–ADP complex, no other differences were observed. We discuss the likely basis of the ability of γ‐phosphate binding to convert preformed GroEL–GroES–ADP–polypeptide complexes into the folding‐active state.

The small heat shock proteins (sHsps) are a key class of molecular chaperones that can inhibit protein aggregation and enhance protein recovery from aggregates. However, the mechanisms sHsps employ to carry out these roles are not well understood, in part because the highly heterogeneous and dynamic particles they form with aggregating proteins are difficult to study with traditional biophysical tools. Here we have applied a novel single particle fluorescence technique known as Burst Analysis Spectroscopy (BAS) to the study of the sHsps IbpA and IbpB (IbpAB). We show that in the presence of IbpAB, two different model proteins converge toward similar, limited aggregate particle size distributions. Additionally, while IbpAB dramatically accelerates the disassembly of protein aggregates by the bacterial KJEB bi-chaperone disaggregase, this enhancement does not appear to be strongly influenced by aggregate particle size. Rather, it is the ability of IbpAB to alter aggregate structure during particle formation that appears to be essential for stimulated disassembly. These observations support a model of aggregate recognition by IbpAB that is not only highly adaptable but capable of shaping aggregate particles into a specialized range of physical properties that are necessary for efficient protein disaggregation.

Fluorescent intercalation complexes of certain polycationic ligands with double-stranded DNA provide a new class of multichromophore labels for fluorescence assays.Fluorescent intercalation complexes of certain polycationic ligands with double-stranded DNA provide a new class of multichromophore labels for fluorescence assays.