S Chakrabarti et al., Molecular chaperones maximize the native state yield on biological times by driving substrates out of equilibrium, National Acad Sciences, 2017.
Molecular chaperones facilitate the folding of proteins and RNA in vivo. Under physiological conditions, the in vitro folding of Tetrahymena ribozyme by the RNA chaperone CYT-19 behaves paradoxically; increasing the chaperone concentration reduces the yield of native ribozymes. In contrast, the protein chap- erone GroEL works as expected; the yield of the native sub- strate increases with chaperone concentration. The discrepant chaperone-assisted ribozyme folding thus contradicts the expec- tation that it operates as an efficient annealing machine. To resolve this paradox, we propose a minimal stochastic model based on the Iterative Annealing Mechanism (IAM) that offers a unified description of chaperone-mediated folding of both pro- teins and RNA. Our theory provides a general relation that quan- titatively predicts how the yield of native states depends on chap- erone concentration. Although the absolute yield of native states decreases in the Tetrahymena ribozyme, the product of the fold- ing rate and the steady-state native yield increases in both cases. By using energy from ATP hydrolysis, both CYT-19 and GroEL drive their substrate concentrations far out of equilibrium, thus maxi- mizing the native yield in a short time. This also holds when the substrate concentration exceeds that of GroEL. Our findings sat- isfy the expectation that proteins and RNA be folded by chaper- ones on biologically relevant time scales, even if the final yield is lower than what equilibrium thermodynamics would dictate. The theory predicts that the quantity of chaperones in vivo has evolved to optimize native state production of the folded states of RNA and proteins in a given time.
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Huafang Xu, Cochaperones enable Hsp70 to use ATP energy to stabilize native proteins out of the folding equilibrium, Scientific Reports, 2018 pp. 1-15.
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Guenter Auernhammer, Instabilities in layered liquids induced by external fields , , 2004 pp. 1-10.
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Anon. (2013), Supplementary Information , , 2013 pp. 1-1.
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D Thirumalai et al., Iterative Annealing Mechanism Explains the Functions of the GroEL and RNA Chaperones, Arxiv.Org, 2019.
Molecular chaperones are ATP-consuming biological machines, which facilitate the folding of proteins and RNA molecules that are kinetically trapped in misfolded states for long times. Unassisted folding occurs by the kinetic partitioning mechanism according to which folding to the native state, with low probability as well as misfolding to one of the many metastable states, with high probability, occur rapidly on similar time scales. GroEL is an all-purpose stochastic machine that assists misfolded substrate proteins (SPs) to fold. The RNA chaperones (CYT-19) help the folding of ribozymes that readily misfold. GroEL does not interact with the folded proteins but CYT-19 disrupts both the folded and misfolded ribozymes. Despite this major difference, the Iterative Annealing Mechanism (IAM) quantitatively explains all the available experimental data for assisted folding of proteins and ribozymes. Driven by ATP binding and hydrolysis and GroES binding, GroEL undergoes a catalytic cycle during which it samples three allosteric states, referred to as T (apo), R (ATP bound), and R'' (ADP bound). In accord with the IAM predictions, analyses of the experimental data shows that the efficiency of the GroEL-GroES machinery and mutants is determined by the resetting rate $k_{R''\rightarrow T}$, which is largest for the wild type GroEL. Generalized IAM accurately predicts the folding kinetics of Tetrahymena ribozyme and its variants. Chaperones maximize the product of the folding rate and the steady state native state fold by driving the substrates out of equilibrium. Neither the absolute yield nor the folding rate is optimized.
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Salvatore Assenza et al., Efficient conversion of chemical energy into mechanical work by Hsp70 chaperones, Arxiv.Org, 2019.
Hsp70 molecular chaperones are abundant ATP-dependent nanomachines that actively reshape non-native, misfolded proteins and assist a wide variety of essential cellular processes. Here we combine complementary computational/theoretical approaches to elucidate the structural and thermodynamic details of the chaperone-induced expansion of a substrate protein, with a particular emphasis on the critical role played by ATP hydrolysis. We first determine the conformational free-energy cost of the substrate expansion due to the binding of multiple chaperones using coarse-grained molecular simulations. We then exploit this result to implement a non-equilibrium rate model which estimates the degree of expansion as a function of the free energy provided by ATP hydrolysis. Our results are in quantitative agreement with recent single-molecule FRET experiments and highlight the stark non-equilibrium nature of the process, showing that Hsp70s are optimized to convert effectively chemical energy into mechanical work close to physiological conditions.
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A Baumketner et al., Effects of Confinement in Chaperonin Assisted Protein Folding: Rate Enhancement by Decreasing the Roughness of the Folding Energy Landscape, Journal Of Molecular Biology, 332 (2003) 701-713.
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Huafeng Xu, Cochaperones enable Hsp70 to fold proteins like a Maxwell's demon, Biorxiv.Org, 2018.
… The copyright holder for this preprint . http://dx.doi.org/ 10.1101 / 283143 doi: bioRxiv preprint first posted online Mar. 15, 2018; Page 2 … The copyright holder for this preprint . http://dx.doi.org/ 10.1101 / 283143 doi: bioRxiv preprint first posted online Mar. 15, 2018; Page 3 …
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Giulia Calloni et al., DnaK Functions as a Central Hub in the E. coli Chaperone Network, Cellreports, 1 (2012) 251-264.
Cellular chaperone networks prevent potentially toxic protein aggregation and ensure proteome integrity. Here, we used Escherichia coli as a model to understand the organization of these networks, focusing on the cooperation of the DnaK system with the upstream chaperone Trigger factor (TF) and the downstream GroEL. Quantitative proteomics revealed that DnaK interacts with at least $700 mostly cytosolic proteins, including $180 relatively aggregation-prone proteins that utilize DnaK exten- sively during and after initial folding. Upon deletion of TF, DnaK interacts increasingly with ribosomal and other small, basic proteins, while its association with large multidomain proteins is reduced. DnaK also functions prominently in stabilizing proteins for subsequent folding by GroEL. These proteins accu- mulate on DnaK upon GroEL depletion and are then degraded, thus defining DnaK as a central organizer of the chaperone network. Combined loss of DnaK and TF causes proteostasis collapse with disruption of GroEL function, defective ribosomal biogenesis, and extensive aggregation of large proteins.
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Kausik Chakraborty et al., Chaperonin-Catalyzed Rescue of Kinetically Trapped States in Protein Folding, Cell, 142 (2010) 112-122.
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Kausik Chakraborty et al., Chaperonin-Catalyzed Rescue of Kinetically Trapped States in Protein Folding, Cell, 142 (2010) 112-122.
GroEL and GroES form a chaperonin nano-cage for single protein molecules to fold in isolation. The folding properties that render a protein chaperonin dependent are not yet understood. Here, we address this question using a double mutant of the maltose- binding protein DM-MBP as a substrate. Upon spon- taneous refolding, DM-MBP populates a kinetically trapped intermediate that is collapsed but structur- ally disordered. Introducing two long-range disulfide bonds into DM-MBP reduces the entropic folding barrier of this intermediate and strongly accelerates native state formation. Strikingly, steric confinement of the protein in the chaperonin cage mimics the kinetic effect of constraining disulfides on folding, in a manner mediated by negative charge clusters in the cage wall. These findings suggest that chaper- onin dependence correlates with the tendency of proteins to populate entropically stabilized folding intermediates. The capacity to rescue proteins from such folding traps may explain the uniquely essential role of chaperonin cages within the cellular chap- erone network.
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Dong-Hua Chen et al., Visualizing GroEL/ES in the Act of Encapsulating a Folding Protein, Cell, 153 (2013) 1354-1365.
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D K Clare et al., Chaperonin complex with a newly folded protein encapsulated in the folding chamber, Nature, 457 (2008) 107-110.
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Jeremy L England and Vijay S Pande, Potential for Modulation of the Hydrophobic Effect Inside Chaperonins, Biophysical Journal, 95 (2008) 3391-3399.
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John M Franck et al., Probing Water Density and Dynamics in the Chaperonin GroEL Cavity, Journal Of The American Chemical Society, 136 (2014) 9396-9403.
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J Frydman, Folding of newly translated proteins in vivo: the role of molecular chaperones., Annual Review Of Biochemistry, 70 (2001) 603-647.
Recent years have witnessed dramatic advances in our understanding of how newly translated proteins fold in the cell and the contribution of molecular chaperones to this process. Folding in the cell must be achieved in a highly crowded macromolecular environment, in which release of nonnative polypeptides into the cytosolic solution might lead to formation of potentially toxic aggregates. Here I review the cellular mechanisms that ensure efficient folding of newly translated proteins in vivo. De novo protein folding appears to occur in a protected environment created by a highly processive chaperone machinery that is directly coupled to translation. Genetic and biochemical analysis shows that several distinct chaperone systems, including Hsp70 and the cylindrical chaperonins, assist the folding of proteins upon translation in the cytosol of both prokaryotic and eukaryotic cells. The cellular chaperone machinery is specifically recruited to bind to ribosomes and protects nascent chains and folding intermediates from nonproductive interactions. In addition, initiation of folding during translation appears to be important for efficient folding of multidomain proteins.
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Florian Georgescauld et al., GroEL/ES Chaperonin Modulates the Mechanism and Accelerates the Rate of TIM-Barrel Domain Folding, Cell, 157 (2014) 922-934.
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F Ulrich Hartl and Manajit Hayer-Hartl, Converging concepts of protein folding in vitro and in vivo, Nature Structural & Molecular Biology, 16 (2009) 574-581.
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F Ulrich Hartl and Manajit Hayer-Hartl, Converging concepts of protein folding in vitro and in vivo, Nature Structural & Molecular Biology, 16 (2009) 574-581.
Most proteins must fold into precise three-dimensional conformations to fulfill their biological functions. Here we review recent concepts emerging from studies of protein folding in vitro and in vivo, with a focus on how proteins navigate the complex folding energy landscape inside cells with the aid of molecular chaperones. Understanding these reactions is also of considerable medical relevance, as the aggregation of misfolding proteins that escape the cellular quality-control machinery underlies a range of debilitating diseases, including many age-onset neurodegenerative disorders.
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F Ulrich Hartl et al., Molecular chaperones in protein folding and proteostasis, Nature, 475 (2011) 324-332.
Most proteins must fold into defined three-dimensional structures to gain functional activity. But in the cellular environ- ment, newly synthesized proteins are at great risk of aberrant folding and aggregation, potentially forming toxic species. To avoid these dangers, cells invest in a complex network of molecular chaperones, which use ingenious mechanisms to prevent aggregation and promote efficient folding. Because protein molecules are highly dynamic, constant chaperone surveillance is required to ensure protein homeostasis (proteostasis). Recent advances suggest that an age-related decline in proteostasis capacity allows the manifestation of various protein-aggregation diseases, including Alzheimer’s disease and Parkinson’s disease. Interventions in these and numerous other pathological states may spring from a detailed under- standing of the pathways underlying proteome maintenance.
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F Ulrich Hartl, Unfolding the chaperone story, Molecular Biology Of The Cell, 28 (2017) 2919-2923.
Protein folding in the cell was originally assumed to be a spontaneous process, based on Anfinsen’s discovery that purified proteins can fold on their own after removal from denaturant. Consequently cell biologists showed little interest in the protein folding process. This changed only in the mid and late 1980s, when the chaperone story began to unfold. As a result, we now know that in vivo, protein folding requires assistance by a complex machinery of molecular chaperones. To ensure efficient folding, members of different chaperone classes receive the nascent protein chain emerging from the ribosome and guide it along an ordered pathway toward the native state. I was fortunate to contribute to these developments early on. In this short essay, I will describe some of the critical steps leading to the current concept of protein folding as a highly organized cellular process.
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H Hofmann et al., Single-molecule spectroscopy of protein folding in a chaperonin cage, National Acad Sciences, 2010.
… Hagen Hofmann, Frank Hillger, Shawn H. Pfeil, Armin Hoffmann, Daniel Streich, Dominik Haenni, Daniel Nettels, Everett A. Lipman, and Benjamin Schuler. PNAS June 29, 2010 107 (26) 11793-11798; https://doi.org/ 10.1073 / pnas . 1002356107 …
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Arthur L Horwich and Wayne A Fenton, Chaperonin-assisted protein folding: a chronologue., Quarterly Reviews Of Biophysics, 2020 32070442, 53 p. e4.
This chronologue seeks to document the discovery and development of an understanding of oligomeric ring protein assemblies known as chaperonins that assist protein folding in the cell. It provides detail regarding genetic, physiologic, biochemical, and biophysical studies of these ATP-utilizing machines from both in vivo and in vitro observations. The chronologue is organized into various topics of physiology and mechanism, for each of which a chronologic order is generally followed. The text is liberally illustrated to provide firsthand inspection of the key pieces of experimental data that propelled this field. Because of the length and depth of this piece, the use of the outline as a guide for selected reading is encouraged, but it should also be of help in pursuing the text in direct order.
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Rahmi Imamoglu et al., Bacterial Hsp70 resolves misfolded states and accelerates productive folding of a multi-domain protein, Nature Communications, 2020 pp. 1-13.
The ATP-dependent Hsp70 chaperones (DnaK in E. coli) mediate protein folding in coop- eration with J proteins and nucleotide exchange factors (E. coli DnaJ and GrpE, respectively). The Hsp70 system prevents protein aggregation and increases folding yields. Whether it also enhances the rate of folding remains unclear. Here we show that DnaK/DnaJ/GrpE accel- erate the folding of the multi-domain protein firefly luciferase (FLuc) ~20-fold over the rate of spontaneous folding measured in the absence of aggregation. Analysis by single-pair FRET and hydrogen/deuterium exchange identified inter-domain misfolding as the cause of slow folding. DnaK binding expands the misfolded region and thereby resolves the kinetically- trapped intermediates, with folding occurring upon GrpE-mediated release. In each round of release DnaK commits a fraction of FLuc to fast folding, circumventing misfolding. We suggest that by resolving misfolding and accelerating productive folding, the bacterial Hsp70 system can maintain proteins in their native states under otherwise denaturing stress conditions.
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Michael J Kerner et al., Proteome-wide Analysis of Chaperonin-Dependent Protein Folding in Escherichia coli, , 122 (2005) 209-220.
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Yujin E Kim et al., Molecular Chaperone Functions in Protein Folding and Proteostasis, Annual Review Of Biochemistry, 82 (2013) 323-355.
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Philipp Koldewey et al., Chaperone-client interactions: Non-specificity engenders multifunctionality, Journal Of Biological Chemistry, 292 (2017) 12010-12017.
Here, we provide an overview of the different mechanisms whereby three different chaperones, Spy, Hsp70, and Hsp60, interact with folding proteins, and we discuss how these chap- erones may guide the folding process. Available evidence sug- gests that even a single chaperone can use many mechanisms to aid in protein folding, most likely due to the need for most chap- erones to bind clients promiscuously. Chaperone mechanism may be better understood by always considering it in the context of the client’s folding pathway and biological function.
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Tom Lopez et al., The Mechanism and Function of Group II Chaperonins, Journal Of Molecular Biology, 427 (2015) 2919-2930.
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Del Lucent et al., Inside the chaperonin toolbox: theoretical and computational models for chaperonin mechanism, Physical Biology, 6 (2009) 015003-9.
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Tania Morán Luengo et al., The Hsp70–Hsp90 Chaperone Cascade in Protein Folding, Trends In Cell Biology, 29 (2019) 164-177.
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Miles S Lyon and Carol Milligan, Extracellular heat shock proteins in neurodegenerative diseases_ New perspectives, Neuroscience Letters, 2019 vol. 711 p. 134462.
ne pathological hallmark of neurodegenerative diseases and CNS trauma is accumulation of insoluble, hy- drophobic molecules and protein aggregations found both within and outside cells. These may be the con- sequences of an inadequate or overburdened cellular response to stresses resulting from potentially toxic changes in extra- and intracellular environments. The upregulated expression of heat shock proteins (HSPs) is one ex- ample of a highly conserved cellular response to both internal and external stress. Intracellularly these proteins act as chaperones, playing vital roles in the folding of nascent polypeptides, the translocation of proteins be- tween subcellular locations, and the disaggregation of misfolded or aggregated proteins in an attempt to maintain cellular proteostasis during both homeostatic and stressful conditions. While the predominant study of the HSPs has focused on their intracellular chaperone functions, it remains unclear if all neuronal populations can mount a complete stress response. Alternately, it is now well established that some members of this family of proteins can be secreted by nearby, non-neuronal cells to act in the extracellular environment. This review addresses the current literature detailing the use of exogenous and extracellular HSPs in the treatment of cellular and animal models of neurodegenerative disease. These findings offer a new measure of therapeutic potential to the HSPs, but obstacles must be overcome before they can be efficiently used in a clinical setting.
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Alireza Mashaghi et al., Chaperone Action at the Single-Molecule Level, Chemical Reviews, 114 (2013) 660-676.
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Fumihiro Motojima, How do chaperonins fold protein?, Biophysics, 11 (2015) 93-102.
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F Motojima and Yuko Motojima-Miyazaki, Revisiting the contribution of negative charges on the chaperonin cage wall to the acceleration of protein folding, National Acad Sciences, 2012.
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Samuel Nkrumah, Protein Folding from the Perspective of Chaperone Action, Arxiv.Org, 2019 1911.11900v1, q-bio.BM.
Predicting the three-dimensional (3D) functional structures of proteins remains an important computational milestone in molecular biology to be achieved. This feat is hinged on a clear understanding of the mechanism which proteins use to fold into their native structures. Since Levinthal's paradox, there has been a lot of progress in understanding this mechanism. Most of the earlier attempts were caught between assigning either hydrophobic interactions or hydrogen bonding as the dominant folding force. However, a consensus now seems to be emerging about hydrogen bonding being a stronger force. Interestingly, a view from chaperone action may further throw some light on the nature of the folding mechanism. Thus the very mechanisms which prevent protein aggregation and misfolding, could help us have a better understanding of the folding mechanism itself.
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Vladimir Reinharz and Tsvi Tlusty, Unspecific binding but specific disruption of the group I intron by the StpA chaperone, Arxiv.Org, 2019 1911.03046v1, q-bio.BM.
Chaperone proteins -- the most disordered among all protein groups -- help RNAs fold into their functional structure by destabilizing misfolded configurations or stabilizing the functional ones. But disentangling the mechanism underlying RNA chaperoning is challenging, mostly due to inherent disorder of the chaperones and the transient nature of their interactions with RNA. In particular, it is unclear how specific the interactions are and what role is played by amino acid charge and polarity patterns. Here, we address these questions in the RNA chaperone StpA. By adapting direct coupling analysis (DCA) to treat in tandem sequences written in two alphabets, nucleotides and amino acids, we could analyze StpA-RNA interactions and identify a two-pronged mechanism: StpA disrupts specific positions in the group I intron while globally and loosely binding to the entire structure. Moreover, the interaction is governed by the charge pattern: negatively charged regions in the destabilizing StpA N-terminal affect a few specific positions in the RNA, located in stems and in the pseudoknot. In contrast, positive regions in the C-terminal contain strongly coupled amino acids that promote non-specific or weakly-specific binding to the RNA. The present study opens new avenues to examine the functions of disordered proteins and to design disruptive proteins based on their charge patterns.
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Ying Ren et al., Explicit solvent molecular dynamics simulations of chaperonin-assisted rhodanese folding, Particuology, 7 (2009) 220-224.
Chaperonins are known to facilitate the productive folding of numerous misfolded proteins. Despite their established importance, the mechanism of chaperonin-assisted protein folding remains unknown. In the present article, all-atom explicit solvent molecular dynamics (MD) simulations have been performed for the first time on rhodanese folding in a series of cavity-size and cavity-charge chaperonin mutants. A compromise between stability and flexibility of chaperonin structure during the substrate folding has been observed and the key factors affecting this dynamic process are discussed.
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S L Rutherford and S Lindquist, Hsp90 as a capacitor for morphological evolution., Nature, 396 (1998) 336-342.
The heat-shock protein Hsp90 supports diverse but specific signal transducers and lies at the interface of several developmental pathways. We report here that when Drosophila Hsp90 is mutant or pharmacologically impaired, phenotypic variation affecting nearly any adult structure is produced, with specific variants depending on the genetic background and occurring both in laboratory strains and in wild populations. Multiple, previously silent, genetic determinants produced these variants and, when enriched by selection, they rapidly became independent of the Hsp90 mutation. Therefore, widespread variation affecting morphogenic pathways exists in nature, but is usually silent; Hsp90 buffers this variation, allowing it to accumulate under neutral conditions. When Hsp90 buffering is compromised, for example by temperature, cryptic variants are expressed and selection can lead to the continued expression of these traits, even when Hsp90 function is restored. This provides a plausible mechanism for promoting evolutionary change in otherwise entrenched developmental processes.
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Helen Saibil, Chaperone machines for protein folding, unfolding and disaggregation, Nature Publishing Group, 14 (2013) 630-642.
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T Saio et al., Oligomerization of a molecular chaperone modulates its activity, Elifesciences.Org, .
Molecular chaperones alter the folding properties of cellular proteins via mechanisms that are not well understood. Here, we show that Trigger Factor (TF), an ATP-independent chaperone, exerts strikingly contrasting effects on the folding of non-native proteins as it transitions between a monomeric and a dimeric state. We used NMR spectroscopy to determine the atomic resolution structure of the 100 kDa dimeric TF. The structural data show that some of the substrate-binding sites are buried in the dimeric interface, explaining …
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Lars Skjærven et al., Dynamics, flexibility, and allostery in molecular chaperonins, Febs Letters, 589 (2015) 2522-2532.
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Fumiko Takagi et al., How protein thermodynamics and folding mechanisms are altered by the chaperonin cage: Molecular simulations, Proceedings Of The National Academy Of Sciences Of The United States Of America, 100 (2003) 11367-11372.
How the Escherichia coli GroEL/ES chaperonin assists folding of a substrate protein remains to be uncovered. Recently, it was suggested that confinement into the chaperonin cage itself can significantly accelerate folding of a substrate. Performing comprehensive molecular simulations of eight proteins confined into various sizes L of chaperonin-like cage, we explore how and to what extent protein thermodynamics and folding mechanisms are altered by the cage. We show that a substrate protein is remarkably stabilized by confinement; the estimated increase in denaturation temperature ΔTf is as large as ≈60°C. For a protein of size R0, the stabilization ΔTf scales as (R0/L)ν, where ν ≈ 3, which is consistent with a mean field theory of polymer. We also found significant free energy cost of confining a protein, which increases with R0/L, indicating that the confinement requires external work provided by the chaperonin system. In kinetic study, we show the folding is accelerated in a modestly well confined case, which is consistent with a recent experimental result on ribulose-1,5-bisphosphate carboxylase-oxygenase folding and simulation results of a β hairpin. Interestingly, the acceleration of folding is likely to be larger for a protein with more complex topology, as quantified by the contact order. We also show how ensemble of folding pathways are altered by the chaperonin-like cage calculating a variant of φ value used in the study of spontaneous folding.
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Yun-Chi Tang et al., Structural features of the GroEL-GroES nano-cage required for rapid folding of encapsulated protein., Cell, 125 (2006) 903-914.
GroEL and GroES form a chaperonin nano-cage for proteins up to approximately 60 kDa to fold in isolation. Here we explored the structural features of the chaperonin cage critical for rapid folding of encapsulated substrates. Modulating the volume of the GroEL central cavity affected folding speed in accordance with confinement theory. Small proteins (approximately 30 kDa) folded more rapidly as the size of the cage was gradually reduced to a point where restriction in space slowed folding dramatically. For larger proteins (approximately 40-50 kDa), either expanding or reducing cage volume decelerated folding. Additionally, interactions with the C-terminal, mildly hydrophobic Gly-Gly-Met repeat sequences of GroEL protruding into the cavity, and repulsion effects from the negatively charged cavity wall were required for rapid folding of some proteins. We suggest that by combining these features, the chaperonin cage provides a physical environment optimized to catalyze the structural annealing of proteins with kinetically complex folding pathways.
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D Thirumalai and George H Lorimer, Chaperonin-mediated protein folding, Annualreviews.Org, .
▪ Abstract Molecular chaperones are required to assist folding of a subset of proteins in Escherichia coli. We describe a conceptual framework for understanding how the GroEL- GroES system assists misfolded proteins to reach their native states. The architecture of …
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D Thirumalai, Caging helps proteins fold, National Acad Sciences, 2003.
How do proteins fold sponta-neously? The quest to answer this question has led to significant developments on theoretical, experimental, and computational fronts in the last decade (1–7). A combination of approaches has provided a detailed understanding of the …
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Xiang Ye et al., Folding of maltose binding protein outside of and in GroEL., Proceedings Of The National Academy Of Sciences Of The United States Of America, 115 (2018) 519-524.
We used hydrogen exchange-mass spectrometry (HX MS) and fluorescence to compare the folding of maltose binding protein (MBP) in free solution and in the GroEL/ES cavity. Upon refolding, MBP initially collapses into a dynamic molten globule-like ensemble, then forms an obligatory on-pathway native-like folding intermediate (1.2 seconds) that brings together sequentially remote segments and then folds globally after a long delay (30 seconds). A single valine to glycine mutation imposes a definable folding defect, slows early intermediate formation by 20-fold, and therefore subsequent global folding by approximately twofold. Simple encapsulation within GroEL repairs the folding defect and reestablishes fast folding, with or without ATP-driven cycling. Further examination exposes the structural mechanism. The early folding intermediate is stabilized by an organized cluster of 24 hydrophobic side chains. The cluster preexists in the collapsed ensemble before the H-bond formation seen by HX MS. The V9G mutation slows folding by disrupting the preintermediate cluster. GroEL restores wild-type folding rates by restabilizing the preintermediate, perhaps by a nonspecific equilibrium compression effect within its tightly confining central cavity. These results reveal an active GroEL function other than previously proposed mechanisms, suggesting that GroEL possesses different functionalities that are able to relieve different folding problems. The discovery of the preintermediate, its mutational destabilization, and its restoration by GroEL encapsulation was made possible by the measurement of a previously unexpected type of low-level HX protection, apparently not dependent on H-bonding, that may be characteristic of proteins in confined spaces.
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