Podcasts about groel es

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.11.08.373423v1?rss=1 Authors: Koculi, E., Thirumalai, D. Abstract: The E. Coli. ATP-consuming chaperonin machinery, a complex between GroEL and GroES, has evolved to facilitate folding of substrate proteins (SPs) that cannot do so spontaneously. A series of kinetic experiments show that the SPs are encapsulated in the GroEL/ES nano cage for a short duration. If confining the SPs in the predominantly polar cage of GroEL in order to help folding, the assisted folding rate, relative to the bulk value, should always be enhanced. Here, we show that this is not the case for the folding of rhodanese in the presence of the full machinery of GroEL/ES and ATP. The assisted folding rate of rhodanese decreases. Based on our finding and those reported in other studies, we suggest that the ATP-consuming chaperonin machinery has evolved to optimize the product of the folding rate and the yield of the folded SPs on the biological time scale. Neither the rate nor the yield is separately maximized. Copy rights belong to original authors. Visit the link for more info

The cylindrical chaperonin GroEL and its lid-shaped cofactor GroES of Escherichia coli perform an essential role in assisting protein folding by transiently encapsulating non-native substrate in an ATP-regulated mechanism. It remains controversial whether the chaperonin system functions solely as an infinite dilution chamber, preventing off-pathway aggregation, or actively enhances folding kinetics by modulating the folding energy landscape. Here we developed single-molecule approaches to distinguish between passive and active chaperonin mechanisms. Using low protein concentrations to exclude aggregation, in combination with highly sensitive spectroscopic methods, such as single-molecule Förster resonance energy transfer (FRET) and fluorescence correlation spectroscopy (FCS), we measured the spontaneous and GroEL/ES-assisted folding of double-mutant maltose binding protein (DM-MBP), and a natural GroEL substrate - dihydrodipicolinate synthase (DapA). We show that both proteins form highly flexible, kinetically trapped folding intermediates, when folding in free solution and do not engage in inter-molecular interactions, such as aggregation, at sufficiently low concentration. We find that in the absence of aggregation, GroEL/ES accelerates folding of DM-MBP up to 8-fold over the spontaneous folding rate. The folding of DapA could be measured at physiological temperature and was found to be ~130-fold accelerated by GroEL/ES. As accelerated folding was independent of repetitive cycles of protein binding and release from GroEL, we demonstrate that iterative annealing does not significantly contribute to chaperonin assisted substrate folding. With a single molecule FRET based approach, we show that a given substrate molecule spends most of the time (~80%) during the GroEL reaction cycle inside the GroEL central cavity, in line with the inner GroEL cage being the active principle in folding catalysis. Moreover, photoinduced electron transfer experiments on DM-MBP provided direct experimental evidence that the confining environment of the chaperonin cage restricts polypeptide chain dynamics. This effect is mainly mediated by the net-negatively charged wall of the GroEL/ES cavity, as shown using the GroEL mutant EL(KKK2) in which the net-negative charge is removed. Taken together, we were able to develop novel approaches, based on single molecule spectroscopy and making use of GroEL as a single molecule sorting machine, to measure GroEL substrate folding rates at sub-nanomolar concentrations. We also, for the first time, provide direct experimental evidence of conformational restriction of an encapsulated polypeptide in a chaperonin cage. Our findings suggest that global encapsulation inside the GroEL/ES cavity, not iterative cycles of annealing and forced unfolding, can accelerate substrate folding by reduction of an entropic energy barrier to the folded state, in strong support of an active chaperonin mechanism. Accelerated folding is biologically significant as it adjusts folding rates relative to the rate of protein synthesis.

The interactome of GroEL/ES has been characterized extensively in several studies and substrates of the chaperonin have been classified (Kerner et al., 2005; Fijiwara et al., 2010). However, the question of what makes some proteins GroEL-dependent and how exactly the chaperonin system promotes their folding remained unresolved. Moreover, it has been unclear how the chaperonin acts on its substrates and whether the protein folding pathway is modified inside the cage as compared to free solution. The aim of this study, therefore, was to characterise and compare the spontaneous and chaperonin-assisted refolding pathway of an obligate substrate of GroEL/ES, in order to elucidate the mechanism of GroEL/ES action. This study presents evidence that encapsulation in the GroEL/ES-cage accelerates the rate and modulates the mechanism of folding of its obligate TIM-barrel substrate, dihydrodipicolinate synthase. We found that the spontaneous refolding of DAPA is slow due to high cooperativity of the process, as it initiates from an ensemble of unstructured intermediates. We demonstrated that the confining environment of the chaperonin cage promotes formation of the TIM-barrel structure in a segmental manner, lowering the entropic component of the activation barrier and accelerating the rate of DAPA folding. Moreover, the spontaneous refolding pathway of a GroEL-independent homolog of DAPA, MsNANA, closely resembles that of DAPA inside the chaperonin cage. Thus, we conclude that GroEL/ES is a powerful folding catalyst for the substrates that otherwise fail to effectively reach their native state.

Folding of newly synthesized proteins is an essential part of protein biosynthesis and misfolding can result in protein aggregation which can also lead to several severe diseases. Protein folding is a highly heterogeneous process and rarely populated intermediate states may play an important role. Single-molecule techniques are ideally suited to resolve these heterogeneities. In this thesis, I have employed a variety of single-molecule fluorescence spectroscopy techniques to study protein folding using model systems on different levels of complexity. The acidic compact state (A state) of Myo- globin is used as a model system of a protein folding intermediate and is studied by a combination of molecular dynamics (MD) simulations and several fluorescence spectroscopic techniques. Using two-focus fluorescence correlation spectroscopy (FCS), it is shown that the A state is less compact than the native state of myoglobin, but not as expanded as the fully unfolded state. The analysis of exposed hydrophobic regions in the acidic structures generated by the MD simulations reveals poten- tial candidates involved in the aggregation processes of myoglobin in the acidic compact state. These results contribute to the understanding of disease-related fibril formation which may lead ultimately to better treatments for these diseases. A huge machinery of specialized proteins, the molecular chaperones, has evolved to assist protein folding in the cell. Using single molecule fluorescence spectroscopy, I have studied several members of this machinery. Single-pair fluorescence resonance energy transfer (spFRET) experiments probed the conformation of the mitochondrial heat shock protein 70 (Hsp70), Ssc1, in different stages along its functional cycle. Ssc1 has a very defined conformation in the ATP state with closely docked domains but shows significantly more heterogeneity in the presence of ADP. This heterogeneity is due to binding and release of ADP. The nucleotide-free state has less inter-domain contacts than the ATP or ADP-bound states. However, the addition of a substrate protein decreases the interaction between the domains even further simultaneously closing the substrate binding lid, showing that substrate binding plays an active role in the remodeling of Ssc1. This behavior is strikingly different than in DnaK, the major bacterial Hsp70. In DnaK, complete domain undocking in the presence of ADP was observed, followed by a slight re-compaction upon substrate binding. These differences may reflect tuning of Ssc1 to meet specific functions, i.e. protein import into mitochondria, in addition to protein folding. Ssc1 requires the assistance of several cofactors depending on the specific task at hand. The results of spFRET experiments suggest that the cofactors modulate the conformation of Ssc1 to enable it to perform tasks as different as protein import and protein folding. Downstream of Hsp70 in the chaperone network, the GroEL/ES complex is a highly specialized molecular machine that is essential for folding of a large subset of proteins. The criteria that distin- guish proteins requiring the assistance of GroEL are not completely understood yet. It is shown here that GroEL plays an active role in the folding of double-mutant maltose binding protein (DM-MBP). DM-MBP assumes a kinetically trapped intermediate state when folding spontaneously, and GroEL rescues DM-MBP by the introduction of entropic constraints. These findings suggest that proteins with a tendency to populate kinetically trapped intermediates require GroEL assistance for folding. The capacity of GroEL to rescue proteins from such folding traps may explain the unique role of GroEL within the cellular chaperone machinery.

Upon emerging from the ribosomal exit tunnel, folding of the polypeptide chain is necessary to form the fully functional protein. In E. coli, correct and efficient protein folding is mainly secured by an organized and complex chaperone system which includes two main principles: The first principle consists of the nascent binding chaperones including trigger factor (TF) and the DnaK/DnaJ system, while the second principle is represented by the downstream GroEL/ES chaperonin system. The identification of ~250 natural GroEL substrates demonstrated that GroEL/ES specifically folds a small group of proteins with complex domain topologies (Kerner et al., 2005) which include some essential proteins. Although the structural, functional and mechanistic aspects of DnaK, the E. coli Hsp70 chaperone, have been extensively studied, a systematic profiling of the natural DnaK substrates is still missing. Moreover, the cooperation between the two main chaperone systems remains to be elucidated. Here we analyzed the central role of DnaK in the bacterial chaperone network and its cooperation with the ribosome-associated chaperone TF and the downstream chaperonin GroEL/GroES using SILAC-based proteomics of DnaK-pulldowns. In parallel, we also analyzed the changes at the global proteome level under conditions of single or combined chaperone deletion. Our measurements show that DnaK normally interacts with at least ~700 newly-synthesized and pre-existent proteins (~30 % of all cytosolic proteins), including ~200 aggregation-prone substrates. Individual deletion of TF or depletion of GroEL/ES at 30 oC-37 oC leads to limited but highly specific changes in the DnaK interactome and in global proteome composition. Specifically, loss of TF results in increased interaction of DnaK with ribosomal and other small, basic proteins, and in a specific defect in the biogenesis of outer membrane -barrel proteins. While deletion of DnaK/DnaJ leads to the degradation or aggregation of ~150 highly DnaK-dependent proteins of large size, massive proteostasis collapse is only observed upon combined deletion of the DnaK system and TF, and is accompanied by extensive aggregation of GroEL substrates and ribosomal proteins. We conclude that DnaK is a central hub in the cytosolic E. coli chaperone network, interfacing with the upstream TF and the downstream chaperonin. These three major chaperone machineries have partially overlapping and non-redundant functions.