Folding of malate dehydrogenase inside the GroEL-GroES cavity

The chaperonin GroEL binds nonnative substrate protein in the hydrophobic central cavity of an open ring. ATP and GroES binding to the same ring converts this cavity into an encapsulated, hydrophilic chamber that mediates productive folding. A 'rack' mechanism of initial protein unfolding proposes that, upon GroES and ATP binding, the polypeptide is stretched between the binding sites on the twisting apical domains of GroEL before complete release into the chamber. Here, the structure of malate dehydrogenase (MDH) subunit during folding is monitored by deuterium exchange, peptic fragment production and mass spectrometry. When bound to GroEL, MDH exhibits a core of partially protected secondary structure that is only modestly deprotected upon ATP and GroES binding. Moreover, deprotection is broadly distributed throughout MDH, suggesting that it results from breaking hydrogen bonds between MDH and the cavity wall or global destabilization, as opposed to forced mechanical unfolding.     

Allosteric signaling of ATP hydrolysis in GroEL-GroES complexes

The double-ring chaperonin GroEL and its lid-like cochaperonin GroES form asymmetric complexes that, in the ATP-bound state, mediate productive folding in a hydrophilic, GroES-encapsulated chamber, the so-called cis cavity. Upon ATP hydrolysis within the cis ring, the asymmetric complex becomes able to accept non-native polypeptides and ATP in the open, trans ring. Here we have examined the structural basis for this allosteric switch in activity by cryo-EM and single-particle image processing. ATP hydrolysis does not change the conformation of the cis ring, but its effects are transmitted through an inter-ring contact and cause domain rotations in the mobile trans ring. These rigid-body movements in the trans ring lead to disruption of its intra-ring contacts, expansion of the entire ring and opening of both the nucleotide pocket and the substrate-binding domains, admitting ATP and new substrate protein.     

Repetitive Protein Unfolding by the trans Ring of the GroEL-GroES Chaperonin Complex Stimulates Folding

A key constraint on the growth of most organisms is the slow and inefficient folding of many essential proteins. To deal with this problem, several diverse families of protein folding machines, known collectively as molecular chaperones, developed early in evolutionary history. The functional role and operational steps of these remarkably complex nanomachines remain subjects of active debate. Here we present evidence that, for the GroEL-GroES chaperonin system, the non-native substrate protein enters the folding cycle on the trans ring of the double-ring GroEL-ATP-GroES complex rather than the ADP-bound complex. The properties of this ATP complex are designed to ensure that non-native substrate protein binds first, followed by ATP and finally GroES. This binding order ensures efficient occupancy of the open GroEL ring and allows for disruption of misfolded structures through two phases of multiaxis unfolding. In this model, repeated cycles of partial unfolding, followed by confinement within the GroEL-GroES chamber, provide the most effective overall mechanism for facilitating the folding of the most stringently dependent GroEL substrate proteins.     

Football- and bullet-shaped GroEL-GroES complexes coexist during the reaction cycle

GroEL is an Escherichia coli chaperonin that is composed of two heptameric rings stacked back-to-back. GroEL assists protein folding with its cochaperonin GroES in an ATP-dependent manner in vitro and in vivo. However, it is still unclear whether GroES binds to both rings of GroEL simultaneously under physiological conditions. In this study, we monitored the GroEL-GroES interaction in the reaction cycle using fluorescence resonance energy transfer. We found that nearly equivalent amounts of symmetric GroEL-(GroES)(2) (football-shaped) complex and asymmetric GroEL-GroES (bullet-shaped) complex coexist during the functional reaction cycle. We also found that D398A, an ATP hydrolysis defective mutant of GroEL, forms a football-shaped complex with ATP bound to the two rings. Furthermore, we showed that ADP prevents the association of ATP to the trans-ring of GroEL, and as a consequence, the second GroES cannot bind to GroEL. Considering the concentrations of ADP and ATP in E. coli, ADP is expected to have a small effect on the inhibition of GroES binding to the trans-ring of GroEL in vivo. These results suggest that we should reconsider the chaperonin-mediated protein-folding mechanism that involves the football-shaped complex.     

Immobilization of invertase by encapsulation in polyelectrolyte complexes.

Free and polystyrene-bound invertase from Saccharomyces cerevisiae were encapsulated within symplex membranes which were composed of cellulose sulfate as the polymeric anion and poly(dimethyldiallylammonium chloride) as the polymeric cation. The kinetics and the performance of the encapsulated enzyme preparations have been compared to the free enzyme employing the hydrolysis of sucrose. The pH and temperature optima were only slightly affected by the encapsulation. The kinetic constants, however, were changed by the encapsulation as a result of diffusional limitation. Encapsulated invertase showed a high storage stability and a high operational stability if low substrate concentrations were applied. The coimmobilization of invertase with living cells, which are not capable of utilizing sucrose, in the described capsules, opens many possibilities in fermentation technology.     

Self-cross-linking polyelectrolyte complexes for therapeutic cell encapsulation

Self-cross-linking polyelectrolytes are used to strengthen the surface of calcium alginate beads for cell encapsulation. Poly([2-(methacryloyloxy)ethyl]trimethylammonium chloride), containing 30 mol % 2-aminoethyl methacrylate, and poly(sodium methacrylate), containing 30 mol % 2-(methacryloyloxy)ethyl acetoacetate, were prepared by radical polymerization. Sequential deposition of these polyelectrolytes on calcium alginate films or beads led to a shell consisting of a covalently cross-linked polyelectrolyte complex that resisted osmotic pressure changes as well as challenges with citrate and high ionic strength. Confocal laser fluorescence microscopy revealed that both polyelectrolytes were concentrated in the outer 7-25 microm of the calcium alginate beads. The thickness of this cross-linked shell increased with exposure time. GPC studies of solutions permeating through analogous flat model membranes showed molecular weight cut-offs between 150 and 200 kg/mol for poly(ethylene glycol), suitable for cell encapsulation. C 2C 12 mouse cells were shown to be viable within calcium alginate capsules coated with the new polyelectrolytes, even though some of the capsules showed fibroid overcoats when implanted in mice due to an immune response.     

Folding and assembly of large macromolecular complexes monitored by hydrogen-deuterium exchange and mass spectrometry

Recent advances in protein mass spectrometry (MS) have enabled determinations of hydrogen deuterium exchange (HDX) in large macromolecular complexes. HDX-MS became a valuable tool to follow protein folding, assembly and aggregation. The methodology has a wide range of applications in biotechnology ranging from quality control for over-expressed proteins and their complexes to screening of potential ligands and inhibitors. This review provides an introduction to protein folding and assembly followed by the principles of HDX and MS detection, and concludes with selected examples of applications that might be of interest to the biotechnology community.     

A dynamic model of long-range conformational adaptations triggered by nucleotide binding in GroEL-GroES

The molecular chaperone, GroEL, essential for correct protein folding in E. coli, is composed of 14 identical subunits organized in two interacting rings, each providing a folding chamber for non‐native substrate proteins. The oligomeric assembly shows positive cooperativity within each ring and negative cooperativity between the rings. Although it is well known that ATP and long‐range allosteric interactions drive the functional cycle of GroEL, an atomic resolution view of how ligand binding modulates conformational adaptations over long distances remains a major challenge. Moreover, little is known on the relation between equilibrium dynamics at physiological temperatures and the allosteric transitions in GroEL. Here we present multiple all‐atom molecular dynamics simulations of the GroEL‐GroES assemblies at different stages of the functional cycle. Combined with an extensive analysis of the complete set of experimentally available structures, principal component analysis and conformer plots, we provide an explicit evaluation of the accessible conformational space of unliganded GroEL. Our results suggest the presence of pre‐existing conformers at the equatorial domain level, and a shift of the conformational ensemble upon ATP‐binding. At the inter‐ring interface the simulations capture a remarkable offset motion of helix D triggered by ATP‐binding to the folding active ring. The reorientation of helix D, previously only observed upon GroES association, correlates with a change of the internal dynamics in the opposite ring. This work contributes to the understanding of the molecular mechanisms in GroEL and highlights the ability of all‐atom MD simulations to model long‐range structural changes and allosteric events in large systems. Proteins 2012;. © 2012 Wiley Periodicals, Inc.     

Probing dynamics and conformational change of the GroEL-GroES complex by 13C NMR spectroscopy

Bacterial chaperonin GroEL with a molecular mass of 800 kDa was studied by (13)C NMR spectroscopy. Carbonyl carbons of GroEL were labeled with (13)C in an amino acid specific manner in order to reduce the number of signals to be observed in the spectrum. Combination of selective labeling and site-directed mutagenesis enabled us to establish the sequence specific assignment of the (13)C resonances from GroEL. ADP-binding induced a chemical shift change of Tyr478 in the equatorial domain and His401 in the intermediate domain, but little of Tyr203 in the apical domain. Upon complex formation with co-chaperonin GroES in the presence of ADP, Tyr478 exhibits two peaks that would originate from the cis and trans rings of the asymmetric GroEL-GroES complex. Comparison between the line width of the GroEL resonances and those from GroES in complex with GroEL revealed broadening disproportionate to the size of GroEL, implying the existence of conformational fluctuations which may be pertinent to the chaperone activity. Based on these results, we concluded that (13)C NMR observation in combination with selective labeling and site-directed mutagenesis can be utilized for probing the conformational change and dynamics of the extremely large molecules that are inaccessible with current NMR methods.     

Folding and catalysis by the VS ribozyme

The VS ribozyme is a 154 nt self-cleaving RNA molecule that can be divided into a trans-acting five-helix ribozyme and stem-loop substrate. The structure of the ribozyme is organised by two three-way helical junctions, the structure of which has been determined by a combination of comparative gel electrophoresis and fluorescence resonance energy transfer experiments. From this, the overall global architecture of the ribozyme has been deduced. The substrate is then thought to dock into the cleft formed between helices II and VI, where it makes a close interaction with the loop containing A730. The A730 loop is the probable active site of the ribozyme, and A756 within it is a strong candidate to play a direct role in the transesterification chemistry, possibly by general acid-base catalysis.     

Asymmetric liposome particles with highly efficient encapsulation of siRNA and without nonspecific cell penetration suitable for target-specific delivery

The discovery of siRNA has been an important step in gene therapy, but the problem of delivering siRNA to a target organ limits its use as a therapeutic drug. Liposomes can be used as a nonviral vector to deliver siRNA to target cells. In this study we developed a novel method of producing asymmetric liposome particles (ALPs) with highly efficient siRNA encapsulation. Two kinds of lipid inverted micelles were prepared for the purpose of obtaining ALPs. The inner one is composed of ionizable cationic 1,2-dioleoyl-3-dimethylammonium-propane (DODAP) and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), which entrap siRNA, and the outer one is composed of 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), DOPE, polyethylene glycol-1,2-distearoyl-sn-glycero-3-phosphatidylethanolamine (PEG-PE), and cholesterol. After mixing the inverted micelles, ALPs encapsulating siRNA were obtained by solvent evaporation and dialysis. This process allowed more than 90% siRNA encapsulation as well as the negatively charged surface. The ALPs protected siRNA from ribonuclease A degradation. ALPs without any surface modification elicited almost no uptake into cells, while the surface-modified ALPs with a polyarginine peptide (R12) induced nonspecific cell penetration. The conjugation of the anti-human epidermal growth factor receptor antibody (anti-EGFR) to ALPs induces an EGFR-mediated uptake into the non-small cell lung cancer cell lines but not into NIH-3T3 cells without the receptor. The siRNA encapsulated in ALPs showed the R12- or anti-EGFR-dependent target gene silencing in NCI-H322 cells. These properties of ALPs are useful for target-specific delivery of siRNA after modification of ALPs with a target-specific ligand.     

Folding specificity induced by loop stiffness

To test the importance of loop stiffness in restricting the heterogeneity of transition state ensemble, we relaxed the distal loop of 10 unstable redesigned hydrophobic core mutants of alpha-spectrin SH3 domain. This was achieved by replacing Asp48 by Gly at the tip of the distal hairpin. Although the change was local in nature, the effect on stabilization was not uniform across the core mutants tested. There is an inverse rough correlation between the stabilization and the increase in buried hydrophobic volume, with respect to the wild type. Interestingly enough, proteins that although unstable are properly folded become molten globule-like after relaxation of the distal loop. These results highlight the importance of stiffness in restricting the conformational heterogeneity of a protein during the folding reaction. An interplay between unspecific hydrophobic interactions and constraint induced by polar interactions, or in this case local stiffness, is essential to achieve a well-ordered folded structure.     

Folding and catalysis by the hairpin ribozyme.

The hairpin ribozyme undergoes a site-specific transesterification cleavage of the phosphodiester backbone. The natural form of the ribozyme is a four-way helical junction, where two arms contain unpaired loops. This folds by pairwise coaxial stacking of helical arms, and a rotation into an antiparallel conformation in which there is close association between the loops. This probably generates the local conformation required to facilitate the trajectory into an in-line SN2 transition state. Folding is induced by the cooperative binding of at least two divalent metal ions, which are probably distributed between the junction and the loop-loop interface. The junction forms the structural scaffold on which the geometry of the ribozyme is built, and structural perturbation of the junction leads to impaired catalytic activity.