Biochemical analysis of a cytosolic small heat shock protein, NtHSP18.3, from Nicotiana tabacum

Small heat shock proteins (sHSPs) are widely distributed, and their function and diversity of structure have been much studied in the field of molecular chaperones. In plants, which frequently have to cope with hostile environments, sHSPs are much more abundant and diverse than in other forms of life. In response to high temperature stress, sHSPs of more than twenty kinds can make up more than 1% of soluble plant proteins. We isolated a genomic clone, NtHSP18.3, from Nicotiana tabacum that encodes the complete open reading frame of a cytosolic class I small heat shock protein. To investigate the function of NtHSP18.3 in vitro, it was overproduced in Escherichia coli and purified. The purified NtHSP18.3 had typical molecular chaperone activity as it protected citrate synthase and luciferase from high temperature-induced aggregation. When E. coli celluar proteins were incubated with NtHSP18.3, a large proportion of the proteins remained soluble at temperatures as high as 70 degrees . Native gel analysis suggested that NtHSP18.3 is a dodecameric oligomer as the form present and showing molecular chaperone activity at the condition tested. Binding of bis-ANS to the oligomers of NtHSP18.3 indicated that exposure of their hydrophobic surfaces increased as the temperature was raised. Taken together, our data suggested that NtHSP18.3 is a molecular chaperone that functions as a dodecameric complex and possibly in a temperature-induced manner.     

ATP-independent thermoprotective activity of Nicotiana tabacum heat shock protein 70 in Escherichia coli

To study the functioning of HSP70 in Escherichia coli, we selected NtHSP70-2 (AY372070) from among three genomic clones isolated in Nicotiana tabacum. Recombinant NtHSP70-2, containing a hexahistidine tag at the amino-terminus, was constructed, expressed in E. coli, and purified by Ni(2+) affinity chromatography and Q Sepharose Fast Flow anion exchange chromatography. The expressed fusion protein, H(6)NtHSP70-2 (hexahistidine-tagged Nicotiana tabacum heat shock protein 70-2), maintained the stability of E. coli proteins up to 90 degrees C. Measuring the light scattering of luciferase (luc) revealed that NtHSP70-2 prevents the aggregation of luc without ATP during high-temperature stress. In a functional bioassay (1 h at 50 degrees C) for recombinant H(6)NtHSP70-2, E. coli cells overexpressing H(6)NtHSP70-2 survived about seven times longer than those lacking H(6)NtHSP70-2. After 2 h at 50 degrees C, only the E. coli overexpressing H(6)NtHSP70-2 survived under such conditions. Our NtHSP70-2 bioassays, as well as in vitro studies, strongly suggest that HSP70 confers thermo-tolerance to E. coli.     

Functional mode of NtHSP17.6, a cytosolic small heat-shock protein fromNicotiana tabacum

Small heat-shock proteins (sHsps) are ubiquitous stress proteins with molecular chaperone activity. They share characteristic homology with the α-crystallin protein of the mammalian eye lens as well as being ATP-independent in their chaperone activity. We isolated a clone for a cytosolic class I sHsp,NtHSP17.6, fromNicotiana tabacum, and analyzed its functional mode for such activity. Following its transformation intoEscherichia coli and its over-expression, NtHSPI 7.6 was purified and examinedin vitro. This purified NtHSPI 7.6 exhibited typical chaperone activity in a light-scattering test. It was enable to protect a model substrate, firefly luciferase, from heat-induced aggregation. Non-denaturing PAGE showed that NtHSP17.6 formed a dodecamer in its native conformation, and was bound to its substrate under heat stress. A labeling test with bis-ANS indicated that this binding might be linked to newly exposed hydrophobic sites of the NtHSPI 7.6 complexes during heat shock. Based on these data, we suggest that NtHSP17.6 is a molecular chaperone that functions as a dodecamer in a heat-induced manner.     

Design of a molecular chaperone-assisted protein folding bioreactor

Escherichia coli molecular chaperone GroEL and co-chaperone GroES are well known to assist the folding/refolding of a diverse range of substrate proteins. Despite this, there have been relatively few reports of the GroEL/GroES molecular chaperone system being used as a biotechnology tool for protein folding/refolding. In this paper, a solution-phase protein folding bioreactor is described that involves the complete GroEL/GroES system. The main features of this bioreactor are the use of a stirred-cell concentrator fitted with a 100 kDa molecular weight cutoff membrane and an attached buffer reservoir. This bioreactor system was used successfully for assisted-batch refolding of guanidinium chloride (Gu-HCl) unfolded mitochondrial malate dehydrogenase (mMDH). We believe that protein folding bioreactor systems of this type could have wide potential utility for the folding/refolding of unfolded protein substrates.     

Structure of human J-type co-chaperone HscB reveals a tetracysteine metal-binding domain

Iron-sulfur proteins play indispensable roles in a broad range of biochemical processes. The biogenesis of iron-sulfur proteins is a complex process that has become a subject of extensive research. The final step of iron-sulfur protein assembly involves transfer of an iron-sulfur cluster from a cluster-donor to a cluster-acceptor protein. This process is facilitated by a specialized chaperone system, which consists of a molecular chaperone from the Hsc70 family and a co-chaperone of the J-domain family. The 3.0 A crystal structure of a human mitochondrial J-type co-chaperone HscB revealed an L-shaped protein that resembles Escherichia coli HscB. The important difference between the two homologs is the presence of an auxiliary metal-binding domain at the N terminus of human HscB that coordinates a metal via the tetracysteine consensus motif CWXCX(9-13)FCXXCXXXQ. The domain is found in HscB homologs from animals and plants as well as in magnetotactic bacteria. The metal-binding site of the domain is structurally similar to that of rubredoxin and several zinc finger proteins containing rubredoxin-like knuckles. The normal mode analysis of HscB revealed that this L-shaped protein preferentially undergoes a scissors-like motion that correlates well with the conformational changes of human HscB observed in the crystals.     

Hsp26: a temperature-regulated chaperone

Small heat shock proteins (sHsps) are a conserved protein family, with members found in all organisms analysed so far. Several sHsps have been shown to exhibit chaperone activity and protect proteins from irreversible aggregation in vitro. Here we show that Hsp26, an sHsp from Saccharomyces cerevisiae, is a temperature-regulated molecular chaperone. Like other sHsps, Hsp26 forms large oligomeric complexes. At heat shock temperatures, however, the 24mer chaperone complex dissociates. Interestingly, chaperone assays performed at different temperatures show that the dissociation of the Hsp26 complex at heat shock temperatures is a prerequisite for efficient chaperone activity. Binding of non-native proteins to dissociated Hsp26 produces large globular assemblies with a structure that appears to be completely reorganized relative to the original Hsp26 oligomers. In this complex one monomer of substrate is bound per Hsp26 dimer. The temperature-dependent dissociation of the large storage form of Hsp26 into a smaller, active species and the subsequent re-association to a defined large chaperone-substrate complex represents a novel mechanism for the functional activation of a molecular chaperone.     

The 2.2 A crystal structure of Hsp33: a heat shock protein with redox-regulated chaperone activity

BACKGROUND: One strategy that cells employ to respond to environmental stresses (temperature, oxidation, and pathogens) is to increase the expression of heat shock proteins necessary to maintain viability. Several heat shock proteins function as molecular chaperones by binding unfolded polypeptides and preventing their irreversible aggregation. Hsp33, a highly conserved bacterial heat shock protein, is a redox-regulated molecular chaperone that appears to protect cells against the lethal effects of oxidative stress. RESULTS: The 2.2 A crystal structure of a truncated E. coli Hsp33 (residues 1-255) reveals a domain-swapped dimer. The core domain of each monomer (1-178) folds with a central helix that is sandwiched between two beta sheets. The carboxyl-terminal region (179-235), which lacks the intact Zn binding domain of Hsp33, folds into three helices that pack on the other subunit. The interface between the two core domains is comprised of conserved residues, including a rare Glu-Glu hydrogen bond across the dyad axis. Two potential polypeptide binding sites that span the dimer are observed: a long groove containing pockets of conserved and hydrophobic residues, and an intersubunit 10-stranded beta sheet "saddle" with a largely uncharged or hydrophobic surface. CONCLUSIONS: Hsp33 is a dimer in the crystal structure. Solution studies confirmed that this dimer reflects the structural changes that occur upon activation of Hsp33 as a molecular chaperone. Patterns of conserved residues and surface charges suggest that two grooves might be potential binding sites for protein folding intermediates.     

Low resolution structure and stability studies of human GrpE#2, a mitochondrial nucleotide exchange factor

GrpE acts as a nucleotide exchange factor for the Hsp70 chaperone system. Only one GrpE isoform is present in Escherichia coli, but for reasons not yet well understood, two GrpE isoforms have been found in mammalian mitochondria.Therefore, studies aimed at evaluating the physico-chemical characteristics of these proteins are important for the comprehension of the function of the Hsp70 chaperone system in different organisms. Here we report biophysical studies on human mitochondrial GrpE isoform 2. Small angle X-ray scattering measurements of human GrpE isoform 2 showed that this protein has a quaternary structure which is similar to those of human GrpE isoform 1 and E. coli GrpE: a dimer with a cruciform elongated shape. However, mitochondrial isoforms differed from each other regarding chemical and thermal denaturation profiles. This fact, combined with results of distinct expression patterns previously reported, point out that these proteins may have different response to external stimuli. Our results also indicate that human GrpE isoform 2 is more similar to the GrpE from E. coli than to human GrpE isoform 1. These results are relevant because differences in the conformation of Hsp70 co-chaperones are considered to be one of the reasons for functional diversity of this system.     

Structural and functional conversion of molecular chaperone ClpB from the gram-positive halophilic lactic acid bacterium Tetragenococcus halophilus mediated by ATP and stress

In this study, we report the purification, initial structural characterization, and functional analysis of the molecular chaperone ClpB from the gram-positive, halophilic lactic acid bacterium Tetragenococcus halophilus. A recombinant T. halophilus ClpB (ClpB(Tha)) was overexpressed in Escherichia coli and purified by affinity chromatography, hydroxyapatite chromatography, and gel filtration chromatography. As demonstrated by gel filtration chromatography, chemical cross-linking with glutaraldehyde, and electron microscopy, ClpB(Tha) forms a homohexameric single-ring structure in the presence of ATP under nonstress conditions. However, under stress conditions, such as high-temperature (>45 degrees C) and high-salt concentrations (>1 M KCl), it dissociated into dimers and monomers, regardless of the presence of ATP. The hexameric ClpB(Tha) reactivated heat-aggregated proteins dependent upon the DnaK system from T. halophilus (KJE(Tha)) and ATP. Interestingly, the mixture of dimer and monomer ClpB(Tha), which was formed under stress conditions, protected substrate proteins from thermal inactivation and aggregation in a manner similar to those of general molecular chaperones. From these results, we hypothesize that ClpB(Tha) forms dimers and monomers to function as a holding chaperone under stress conditions, whereas it forms a hexamer ring to function as a disaggregating chaperone in cooperation with KJE(Tha) and ATP under poststress conditions.     

Over-expression of tobacco NtHSP70-1 contributes to drought-stress tolerance in plants

HSP70, a heat shock protein, is a molecular chaperone responsive to various environmental stresses. Here, NtHSP70-1 (AY372069) was a drought-/ABA-inducible gene. We monitored the expression of CaERD15 (early responsive to dehydration, DQ267932) with exposing plants to progressive drought stress. Its activity was used as an indicator of water-deficit conditions. To analyze the protective role of HSP70, we obtained transgenic tobacco plants that constitutively expressed elevated levels of the tobacco HSP70, NtHSP70-1, as well as transgenic plants containing either the vector alone or else having NtHSP70-1 in the antisense orientation. Plants with enhanced levels of NtHSP70-1 in their transgenic sense lines exhibited tolerance to water stress. Under progressive drought, the amount of leaf NtHSP70-1 was correlated with maintenance of optimum water content, with contents being higher in the leaves of dehydrated transgenic sense plants than in those of either the control (vector-only) or the transgenic antisense plants. Moreover, the expression of CaERD15 was considerably reduced in tobacco plants that over-expressed NtHSP70-1. These results suggest that elevated levels of NtHSP70-1 can confer drought-stress tolerance.     

DsbG, a protein disulfide isomerase with chaperone activity

DsbG, a protein disulfide isomerase present in the periplasm of Escherichia coli, is shown to function as a molecular chaperone. Stoichiometric amounts of DsbG are sufficient to prevent the thermal aggregation of two classical chaperone substrate proteins, citrate synthase and luciferase. DsbG was also shown to interact with refolding intermediates of chemically denatured citrate synthase and prevents their aggregation in vitro. Citrate synthase reactivation experiments in the presence of DsbG suggest that DsbG binds with high affinity to early unstructured protein folding intermediates. DsbG is one of the first periplasmic proteins shown to have general chaperone activity. This ability to chaperone protein folding is likely to increase the effectiveness of DsbG as a protein disulfide isomerase.     

Domain Organization of the Monomeric Form of the Tom70 Mitochondrial Import Receptor

Tom70 is a mitochondrial protein import receptor composed of 11 tetratricopeptide repeats (TPRs). The first three TPRs form an N-terminal domain that recruits heat shock protein family chaperones, while the eight C-terminal TPRs form a domain that receives, from the bound chaperone, mitochondrial precursor proteins destined for import. Analytical ultracentrifugation and solution small-angle X-ray scattering (SAXS) analysis characterized Tom70 as an elongated monomer. A model for the Tom70 monomer was proposed based on the alternate interpretation of the domain pairings observed in the crystal structure of the Tom70 dimer and refined against the SAXS data. In this “open” model of the Tom70 monomer, the chaperone- and precursor-binding sites are exposed and lay side by side on one face of the molecule. Fluorescence anisotropy measurements indicated that monomeric Tom70 can bind both chaperone and precursor peptides and that chaperone peptide binding does not alter the affinity of Tom70 for the precursor peptide. SAXS was unable to detect any shape change in Tom70 upon chaperone binding. However, molecular modeling indicated that chaperone binding is incompatible with Tom70 dimer formation. It is proposed that the Tom70 monomer is the functional unit mediating initial chaperone docking and precursor recognition.     

Chaperone-like activity of the N-terminal region of a human small heat shock protein and chaperone-functionalized nanoparticles

Small heat shock proteins (sHsps) are molecular chaperones employed to interact with a diverse range of substrates as the first line of defense against cellular protein aggregation. The N-terminal region (NTR) is implicated in defining features of sHsps; notably in their ability to form dynamic and polydisperse oligomers, and chaperone activity. The physiological relevance of oligomerization and chemical-scale mode(s) of chaperone function remain undefined. We present novel chemical tools to investigate chaperone activity and substrate specificity of human HspB1 (B1NTR), through isolation of B1NTR and development of peptide-conjugated gold nanoparticles (AuNPs). We demonstrate that B1NTR exhibits chaperone capacity for some substrates, determined by anti-aggregation assays and size-exclusion chromatography. The importance of protein dynamics and multivalency on chaperone capacity was investigated using B1NTR-conjugated AuNPs, which exhibit concentration-dependent chaperone activity for some substrates. Our results implicate sHsp NTRs in chaperone activity, and demonstrate the therapeutic potential of sHsp-AuNPs in rescuing aberrant protein aggregation.     

Clusterin is an ATP-independent chaperone with very broad substrate specificity that stabilizes stressed proteins in a folding-competent state

We recently reported that the ubiquitous, secreted protein clusterin has chaperone activity in vitro [Humphreys et al. (1999) J. Biol. Chem. 274, 6875-6881]. In this study, we demonstrate that clusterin (i) inhibits stress-induced precipitation of a very broad range of structurally divergent protein substrates, (ii) binds irreversibly via an ATP-independent mechanism to stressed proteins to form solubilized high molecular weight complexes, (iii) lacks detectable ATPase activity, (iv) when acting alone, does not effect refolding of stressed proteins in vitro, and (v) stabilizes stressed proteins in a state competent for refolding by heat shock protein 70 (HSP70). Furthermore, we show that, at physiological levels, clusterin inhibits stress-induced precipitation of proteins in undiluted human serum. Clusterin represents the first identified secreted mammalian chaperone. However, reports from others suggest that, at least under stress conditions, clusterin may be retained within cells to exert a protective effect. Regardless of the topological site(s) of action, the demonstration that clusterin can stabilize stressed proteins in a refolding-competent state suggests that, during stresses, the action of clusterin may inhibit rapid and irreversible protein precipitation and produce a reservoir of inactive but stabilized molecules from which other refolding chaperones can subsequently salvage functional proteins.     

Characterization of the Escherichia coli YedU protein as a molecular chaperone

We have cloned, purified to homogeneity, and characterized as a molecular chaperone the Escherichia coli YedU protein. The purified protein shows a single band at 31 kDa on SDS-polyacrylamide gels and forms dimers in solution. Like other chaperones, YedU interacts with unfolded and denatured proteins. It promotes the functional folding of citrate synthase and alpha-glucosidase after urea denaturation and prevents the aggregation of citrate synthase under heat shock conditions. YedU forms complexes with the permanently unfolded protein, reduced carboxymethyl alpha-lactalbumin. In contrast to DnaK/Hsp70, ATP does not stimulate YedU-dependent citrate synthase renaturation and does not affect the interaction between YedU and unfolded proteins, and YedU does not display any peptide-stimulated ATPase activity. We conclude that YedU is a novel chaperone which functions independently of an ATP/ADP cycle.     

Localization of GroEL determined by in vivo incorporation of a fluorescent amino acid

The molecular chaperone GroEL is required for bacterial growth under all conditions, mediating folding assistance, via its central cavity, to a diverse set of cytosolic proteins; yet the subcellular localization of GroEL remains unresolved. An earlier study, using antibody probing of fixed Escherichia coli cells, indicated colocalization with the cell division protein FtsZ at the cleavage furrow, while a second E. coli study of fixed cells indicated more even distribution throughout the cytoplasm. Here, for the first time, we have examined the spatial distribution of GroEL in living cells using incorporation of a fluorescent unnatural amino acid into the chaperone. Fluorescence microscopy indicated that GroEL is diffusely distributed, both under normal and stress conditions. Importantly, the present procedure uses a small, fluorescent unnatural amino acid to visualize GroEL in vivo, avoiding the steric demands of a fluorescent protein fusion, which compromises proper GroEL assembly. Further, this unnatural amino acid incorporation avoids artifacts that can occur with fixation and antibody staining.     

Structural and functional characterization of Helicobacter pylori DsbG

Dsb proteins play important roles in bacterial pathogenicity. To better understand the role of Dsb proteins in Helicobacter pylori, we have structurally and functionally characterized H. pylori DsbG (HP0231). The monomer consists of two domains connected by a helical linker. Two monomers associate to form a V-shaped dimer. The monomeric and dimeric structures of H. pylori DsbG show significant differences compared to Escherichia coli DsbG. Two polyethylene glycol molecules are bound in the cleft of the V-shaped dimer, suggesting a possible role as a chaperone. Furthermore, we show that H. pylori DsbG functions as a reductase against HP0518, a putative L,D-transpeptidase with a catalytic cysteine residue.     

Overexpression and protein folding of a chimeric beta-glucosidase constructed from Agrobacterium tumefaciens and Cellvibrio gilvus

In continuation of our investigation on structure and function relationship of beta-glucosidases from mesophilic and thermophilic bacteria, we constructed a chimeric gene by shuffling 17% length in C terminal region of beta-glucosidase of Agrobacterium tumefaciens with the corresponding homologous region of Cellvibrio gilvus beta-glucosidase. The chimeric gene was overexpressed in E. coli BL21 (DE3) using pET vector. However, nearly all of the beta-glucosidase produced was trapped into inclusion bodies in catalytically non-functional state. Attempts were made to solubilize the overexpressed protein by co-expression with molecular chaperone, GroEL/ES, in vivo. The molecular chaperone assisted protein folding that had earlier yielded encouraging results, did not improve the solubilization in the present case with a chimeric beta-glucosidase. Further, we explored protein renaturation under in vitro conditions using various dialysis strategies. Dialysis, rapid dilution and a newly devised method of folding immobilized proteins yielded active enzyme. The usefulness of the in vitro folding methods to obtain functional enzymes from overproduced but non-functional proteins has been discussed.