80 research outputs found

    Biochemical characterizations of Escherichia coli DnaK and DnaK mutant proteins purified from ndk deficient cells

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    Thesis (Ph.D.)--Massachusetts Institute of Technology, Dept. of Biology, 1999.Includes bibliographical references.by Thomas K. Barthel.Ph.D

    A NMR Investigation of Structure and Allostery in the Hsp70 Chaperone System.

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    The Hsp70 Chaperone system is a complex molecular machine composed of proteins. It is responsible for assisting in protein folding/refolding/processing and transport. Hsp70 proteins have been historically associated with the ‘stress’ or ‘heatshock’ response of cells. They function in association with several co-chaperones in a delicately balanced and regulated two stage functional cycle. This system and its component molecules has been the subject of intensive efforts in structural biophysics for the last two decades. In this dissertation we present an analysis of the allosteric machinery, which operates in this molecular system. Using residual dipolar coupling analysis, a state-of-the-art method in solution nuclear magnetic resonance (NMR) spectroscopy, we have been able to detect distinct changes in the T.th-DnaK (the thermophilic Hsp70 protein) as the protein switches between the different stages of its ‘two-stroke’ functional cycle. Specifically, we have seen an opening of the nucleotide binding cleft in the ADP.PI state, without any exchange factor involved. Furthermore, we observed an opening of the IA/IIA interface cleft which would account for the linker being structured in the ATP state and explain how the interdomain allostery works in Hsp70s. Such changes have been observed for the first time in this field. We have also refined new methods to detect residual dipolar couplings and scrutinized our results with the most stringent possible statistical self-validation analysis. In the second stage of this dissertation, we have studied the interaction of E.coli DnaK (the bacterial Hsp70 molecule) with DnaJ (the equivalent bacterial co-chaperone) using paramagnetic relaxation enhancement in solution NMR. Based on this analysis, we propose a model for the interaction of DnaK with DnaJ, where DnaJ lies transversely across both domains of DnaK and might act as a ‘molecular crowbar’ in wedging the two domains apart. This study sheds new light on how the DnaJ co-chaperone system structurally interacts with the primary Hsp70 system and suggests a possible mechanical model which explains how the allosteric machinery operates in the complex.Ph.D.BiophysicsUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/75889/1/akashb_1.pd

    Assembly and biochemical properties of a human chaperone/co-chaperone protein complex

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    The 70kDa members of the heat shock protein family (eg. Hsp70) function as molecular chaperones by binding to exposed hydrophobic patches on nascent polypeptides forming non-covalent interactions, thereby preventing their aggregation and facilitating their proper folding. The folding reaction comprises of cyclic binding and release of the unfolded substrate powered by ATP hydrolysis. Hsp70 requires the assistance of a co-chaperone, generally provided by the Hsp40 group of proteins, for the cycle of protein folding. Biochemical analyses have mapped the possible sites of interaction between the Hsp70 and Hsp40 proteins and predicted a bipartite mode of interaction between Hsp70 and Hsp40. However, structural investigations into the mechanistic features of the folding cycle have been hampered by the transient nature of interaction. The underlying theme for this work was to therefore structurally understand the assembly of Hsp70 proteins with the Hsp40 co-chaperones as a complex during the Hsp70-assisted folding cycle. This study was carried out using human Hsc70 and HSJ1b as representatives of the Hsp70 and Hsp40 families respectively. The first step to understand this co-operation was to develop a strategy to isolate a complex of Hsc70 and HSJ1b suitable for structural studies. Previous studies have reconstituted the Hsp70/Hsp40 complex in vitro by combining the two proteins in molar ratios in the presence of ATP. In this work a co-expression system was developed and a recombinant form of the human Hsc70/HSJ1b complex was successfully purified using a bacterial expression system. Biochemical characterisation revealed that this chaperone complex can protect ~85% of substrate protein from thermal aggregation. Gel filtration analysis revealed that the complex was composed of a heterogenous mix of ~220 kDa and hetero-oligomeric co-polymer species. Analytical ultracentrifugation confirmed that these hetero-oligomeric co-polymer species were not aggregates, and molecular weight for this species was estimated to be 1.1 MDa. These two species represent potentially two different states of association between Hsc70 and HSJ1b. ATP and heat treatment at 42oC with luciferase were identified as factors which promote the conversion of the oligomeric Hsc70/HSJ1b species to the ~220 kDa Hsc70/HSJ1b species. Domain variants of Hsc70 were then generated and their ability to complex with HSJ1b was investigated. Using these Hsc70 domain variants, the region on Hsc70 paramount for polymerisation was identified. The C-terminal 10 kDa lid region was found to be essential for the chaperone/co-chaperone interaction, since the removal of this zone alters binding, function and conformational properties of the Hsc70 and HSJ1b interaction. X-ray crystallography studies on the full length and the domain complexes were carried out, leading to the structure of the apo form of the nucleotide binding domain of Hsc70. Preliminary electron microscopy (EM) analysis was undertaken of the recombinant Hsc70/HSJ1b complex. The preliminary results from negative staining revealed mostly circular particles and were extremely encouraging. Currently work is being carried out to improve sample homogeneity, which will facilitate further EM studies. Thus the recombinant complex generated in this study is an attractive tool to further our understanding of the functional and structural features of the interactions of Hsp70 with Hsp40.EThOS - Electronic Theses Online ServiceGBUnited Kingdo

    Assembly and biochemical properties of a human chaperone/co-chaperone protein complex

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    The 70kDa members of the heat shock protein family (eg. Hsp70) function as molecular chaperones by binding to exposed hydrophobic patches on nascent polypeptides forming non-covalent interactions, thereby preventing their aggregation and facilitating their proper folding. The folding reaction comprises of cyclic binding and release of the unfolded substrate powered by ATP hydrolysis. Hsp70 requires the assistance of a co-chaperone, generally provided by the Hsp40 group of proteins, for the cycle of protein folding. Biochemical analyses have mapped the possible sites of interaction between the Hsp70 and Hsp40 proteins and predicted a bipartite mode of interaction between Hsp70 and Hsp40. However, structural investigations into the mechanistic features of the folding cycle have been hampered by the transient nature of interaction. The underlying theme for this work was to therefore structurally understand the assembly of Hsp70 proteins with the Hsp40 co-chaperones as a complex during the Hsp70-assisted folding cycle. This study was carried out using human Hsc70 and HSJ1b as representatives of the Hsp70 and Hsp40 families respectively. The first step to understand this co-operation was to develop a strategy to isolate a complex of Hsc70 and HSJ1b suitable for structural studies. Previous studies have reconstituted the Hsp70/Hsp40 complex in vitro by combining the two proteins in molar ratios in the presence of ATP. In this work a co-expression system was developed and a recombinant form of the human Hsc70/HSJ1b complex was successfully purified using a bacterial expression system. Biochemical characterisation revealed that this chaperone complex can protect ~85% of substrate protein from thermal aggregation. Gel filtration analysis revealed that the complex was composed of a heterogenous mix of ~220 kDa and hetero-oligomeric co-polymer species. Analytical ultracentrifugation confirmed that these hetero-oligomeric co-polymer species were not aggregates, and molecular weight for this species was estimated to be 1.1 MDa. These two species represent potentially two different states of association between Hsc70 and HSJ1b. ATP and heat treatment at 42oC with luciferase were identified as factors which promote the conversion of the oligomeric Hsc70/HSJ1b species to the ~220 kDa Hsc70/HSJ1b species. Domain variants of Hsc70 were then generated and their ability to complex with HSJ1b was investigated. Using these Hsc70 domain variants, the region on Hsc70 paramount for polymerisation was identified. The C-terminal 10 kDa lid region was found to be essential for the chaperone/co-chaperone interaction, since the removal of this zone alters binding, function and conformational properties of the Hsc70 and HSJ1b interaction. X-ray crystallography studies on the full length and the domain complexes were carried out, leading to the structure of the apo form of the nucleotide binding domain of Hsc70. Preliminary electron microscopy (EM) analysis was undertaken of the recombinant Hsc70/HSJ1b complex. The preliminary results from negative staining revealed mostly circular particles and were extremely encouraging. Currently work is being carried out to improve sample homogeneity, which will facilitate further EM studies. Thus the recombinant complex generated in this study is an attractive tool to further our understanding of the functional and structural features of the interactions of Hsp70 with Hsp40

    Strategies for Modulating the Diverse Activities of Heat Shock Protein 70.

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    Heat shock protein 70 (Hsp70) is an essential regulator of protein homeostasis. Dysfunction of protein homeostasis is directly linked to many diseases, including cancer and neurodegeneration. Thus, an understanding of Hsp70’s roles in this process is expected to provide insights into the mechanisms of disease and, potentially, provide new opportunities for therapies. However, Hsp70 is also involved in essential cellular functions, so it is not clear how to safely target it. In this thesis, I first review how Hsp70 cooperates with co-chaperones to enable its many activities. Hsp70 binds to distinct co-chaperones to form complexes that have individual functions in protein folding, degradation and trafficking, suggesting that inhibition of the protein-protein interactions (PPIs) between Hsp70 and its co-chaperones might be one promising way to safely modulate this system. In Chapter 2, I performed a comprehensive, comparative study on how five TPR domain-containing co-chaperones bind to Hsp70 in vitro. These experiments highlighted the opportunities and challenges of targeting this PPI. In Chapter 3, I demonstrate how allosteric networks in Hsp70 can be manipulated, using both chemical and genetic approaches, in order to regulate binding to co-chaperones and tune chaperone activity in unexpected ways. Taking all this information together, I show in Chapter 4 that allosteric inhibitors of Hsp70 have surprisingly potent antibiotic activity in drug-resistant bacteria, which seem to rely on robust protein homeostasis. By better understanding allostery and PPIs in the Hsp70 network, I made new insights into Hsp70 biology and also discovered new lead compounds for therapeutic development.PHDChemical BiologyUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/116624/1/vaa_1.pd

    Characterization of chloroplast transit peptides and the major stromal Hsp70, CSS1 : implications for an ATP-dependent chloroplast protein import molecular motor

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    Chloroplast protein import is a relatively poorly understood protein trafficking system. In other protein import systems, the translocation machinery has been identified and well studied, including the mechanism by which proteins are unidirectionally transported across the importing membrane. In the chloroplast, no such molecular motor is acknowledged. The work described in this dissertation is a preliminary attempt to assign that role to the major stromal Hsp70, CSS1. We have shown, through a variety of in vivo and in vitro techniques, interaction between a chloroplast transit peptide and two members of the Hsp70 class of molecular chaperones, DnaK and CSS1. We have also mapped this specific interaction to the N-terminus of one transit peptide and generallized this N-terminal bias to all transit peptides through statistical analyses. Futhermore, we have generated a recombinant form of CSS1 and have developed a novel chromatographic technique to purify it in an active form. Finally, we have biochemically characterized CSS1, relating its place within the Hsp70 protein family and describing its catalytic and chaperone activities in detail. This work provides the basis for further in vivo and in vitro studies which our data predict will prove that CSS1 is the chloroplast protein import molecular motor

    Chaperone activation of the hepadnaviral reverse transcriptase for template RNA binding is established by the Hsp70 and stimulated by the Hsp90 system

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    Hepadnaviruses are DNA viruses that replicate by protein-primed reverse transcription, employing a specialized reverse transcriptase (RT), P protein. DNA synthesis from the pregenomic RNA is initiated by binding of P to the Δ signal. Using Δ as template and a Tyr-residue for initiation, the RT synthesizes a DNA oligo (priming) as primer for full-length DNA. Priming strictly requires prior RT activation by chaperones. Active P–Δ complexes have been reconstituted in vitro, but whether in addition to the heat-shock protein 70 (Hsp70) system the Hsp90 system is essential has been controversial. Here we quantitatively compared Hsp70 versus Hsp70 plus Hsp90 RT activation, and corroborated that the Hsp70 system alone is sufficient; however, Hsp90 as well the Hsp70 nucleotide exchange factor Bag-1 markedly stimulated activation by increasing the steady-state concentration of the activated metastable RT form P*, though by different mechanisms. Hsp90 inhibition in intact cells by geldanamycin analogs blocked hepadnavirus replication, however not completely and only at severely cytotoxic inhibitor concentrations. While compatible with a basal level of Hsp90 independent in vivo replication, unambiguous statements are precluded by the simultaneous massive upregulation of Hsp70 and Hsp90

    High Throughput Screens Against Heat Shock Protein 70 (Hsp 70)

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    The molecular chaperone Hsp70 plays important roles in protein quality control. Moreover, Hsp70 has been linked to diseases of protein misfolding, such as neurodegenerative disorders, suggesting that it could be a promising new drug target. However, the molecular mechanisms that link Hsp70 to these diseases remain unclear. We hypothesized that one way to better understand the roles of Hsp70 would be to develop chemical probes that disrupt its specific functions. In this thesis, we developed the first high throughput screens for the ATPase activity of Hsp70. Using this platform, we explored the idea of “gray-box” screening, in which multiple components of the Hsp70 chaperone system are reconstituted in vitro to better approximate the biochemical properties of the physiological complexes. We wanted to understand whether this approach would provide a compromise between “black box” cell-based screens and assays that rely on individual purified proteins. Accordingly, we screened over 50,000 compounds and natural product extracts and reported a number of new Hsp70 inhibitors, including myricetin. Interestingly, we found that many of these inhibitors blocked the protein-protein interactions between the Hsp70, DnaK, and its important co-chaperone, DnaJ. Thus, we expect these compounds to be powerful probes for exploring the biological roles of the DnaK-DnaJ complex. Finally, we explored whether other Hsp70 chaperone functions, such as substrate binding or refolding, might also be useful targets for high throughput screening. Towards that goal, we generated point mutants in DnaK and human Hsc70 and studied how their different in vitro biochemical activities correlated with cellular functions. We found that luciferase refolding activity, not ATPase rate, was more predictive of certain cellular chaperone activities, such as heat shock rescue and effects on tau stability. These results suggest the need for multiple primary and secondary assays in searching for Hsp70 inhibitors. Together, these studies have provided important insights into the Hsp70 chaperone system and they have discovered molecules that could be used to further validate Hsp70 as a drug target.Ph.D.Chemical BiologyUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/86340/1/taiwlyra_1.pd
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