Cooperative unfolding of apolipoprotein A-1 induced by chemical denaturation.

Apolipoprotein A-1 (Apo A-1) plays an important role in lipid transfer and obesity. Chemical unfolding of α-helical Apo A-1 is induced with guanidineHCl and monitored with differential scanning calorimetry (DSC) and CD spectroscopy. The unfolding enthalpy and the midpoint temperature of unfolding decrease linearly with increasing guanidineHCl concentration, caused by the weak binding of denaturant. At room temperature, binding of 50-60 molecules guanidineHCl leads to a complete Apo A-1 unfolding. The entropy of unfolding decreases to a lesser extent than the unfolding enthalpy. Apo A-1 chemical unfolding is a dynamic multi-state equilibrium that is analysed with the Zimm-Bragg theory modified for chemical unfolding. The chemical Zimm-Bragg theory predicts the denaturant binding constant KD and the protein cooperativity σ. Chemical unfolding of Apo A-1 is two orders of magnitude less cooperative than thermal unfolding. The free energy of thermal unfolding is ~0.2 kcal/mol per amino acid residue and ~1.0 kcal/mol for chemical unfolding at room temperature. The Zimm-Bragg theory calculates conformational probabilities and the chemical Zimm-Bragg theory predicts stretches of α-helical segments in dynamic equilibrium, unfolding and refolding independently and fast.

Thermodynamics of protein self-association and unfolding. The case of apolipoprotein A-I.

Protein self-association and protein unfolding are two temperature-dependent processes whose understanding is of utmost importance for the development of biological pharmaceuticals because protein association may stabilize or destabilize protein structure and function. Here we present new theoretical and experimental methods for analyzing the thermodynamics of self-association and unfolding. We used isothermal dilution calorimetry and analytical ultracentrifugation to measure protein self-association and introduced binding partition functions to analyze the cooperative association equilibria. In a second type of experiment, we monitored thermal protein unfolding with differential scanning calorimetry and circular dichroism spectroscopy and used the Zimm−Bragg theory to analyze the unfolding process. For α-helical proteins, the cooperative Zimm−Bragg theory appears to be a powerful alternative to the classical two-state model. As a model protein, we chose highly purified human recombinant apolipoprotein A-I. Self-association of Apo A-I showed a maximum at 21 °C with an association constant Ka of 5.6 × 10(5) M(−1), a cooperativity parameter σ of 0.003, and a maximal association number n of 8. The association enthalpy was linearly dependent on temperature and changed from endothermic at low temperatures to exothermic above 21 °C with a molar heat capacity ΔC(p)° of −2.76 kJ mol(−1) K(−1). Above 45 °C, the association could no longer be measured because of the onset of unfolding. Unfolding occurred between 45 and 65 °C and was reversible and independent of protein concentration up to 160 µM. The midpoint of unfolding (T(0)) as measured by DSC was 52−53 °C; the enthalpy of unfolding (ΔH(N)(U)) was 420 kJ/mol. The molar heat capacity (Δ(N)(U)C(p)) increased by 5.0 ± 0.5 kJ mol(−1) K(−1) upon unfolding corresponding to a loss of 80−85 helical segments, which was confirmed by circular dichroism spectroscopy. Unfolding was highly cooperative with a nucleation parameter σ of 4.4 × 10(−5).

Glutamine synthetase isolated from human brain: octameric structure and homology of partial primary structure with human liver glutamine synthetase.

Glutamine synthetase (GS) has been purified from the cytosolic fraction of non-frozen human brain tissue. The purified GS migrated as a main band around 44 kD on reducing SDS-PAGE. Two-dimensional electrophoresis revealed heterogeneity within subunits of GS. The masses of eight different peptides from a tryptic digest of GS as measured by high resolution MALDI-MS matched with the respective masses from an in silico tryptic fingerprint of the Swiss-Prot database entry of human liver GS, proving that at least 24% of the primary sequences of GS from brain and liver are identical. Sedimentation equilibrium profiles obtained from analytical ultracentrifugation experiments at 10 degrees C showed that human brain GS is mainly octameric. The quaternary structure of human brain GS at 10 microM (subunit concentration) was not significantly affected by cations, such as magnesium (5 and 20 mM) or manganese (0.2 and 1 mM) within the range of pH 7.1-7.8.

Reversible thermal transition in GrpE, the nucleotide exchange factor of the DnaK heat-shock system.

DnaK, a Hsp70 acting in concert with its co-chaperones DnaJ and GrpE, is essential for Escherichia coli to survive environmental stress, including exposure to elevated temperatures. Here we explored the influence of temperature on the structure of the individual components and the functional properties of the chaperone system. GrpE undergoes extensive but fully reversible conformational changes in the physiologically relevant temperature range (transition midpoint at approximately 48 degrees C), as observed with both circular dichroism measurements and differential scanning calorimetry, whereas no thermal transitions occur in DnaK and DnaJ between 15 degrees C and 48 degrees C. The conformational changes in GrpE appear to be important in controlling the interconversion of T-state DnaK (ATP-liganded, low affinity for polypeptide substrates) and R-state DnaK (ADP-liganded, high affinity for polypeptide substrates). The rate of the T --> R conversion of DnaK due to DnaJ-triggered ATP hydrolysis follows an Arrhenius temperature dependence. In contrast, the rate of the R --> T conversion due to GrpE-catalyzed ADP/ATP exchange increases progressively less with increasing temperature and even decreases at temperatures above approximately 40 degrees C, indicating a temperature-dependent reversible inactivation of GrpE. At heat-shock temperatures, the reversible structural changes of GrpE thus shift DnaK toward its high-affinity R state.

The HspR regulon of Streptomyces coelicolor: a role for the DnaK chaperone as a transcriptional co-repressordagger.

The dnaK operon of Streptomyces coelicolor encodes the DnaK chaperone machine and HspR, the transcriptional repressor of the operon; HspR confers repression by binding to several inverted repeat sequences in the promoter region, dnaKp. Here, we demonstrate that HspR specifically requires the presence of DnaK protein to retard a dnaKp fragment in gel-shift assays. This requirement is independent of the co-chaperones, DnaJ and GrpE, and it is ATP independent. Furthermore the retarded protein-DNA complex can be 'supershifted' by anti-DnaK monoclonal antibody, demonstrating that DnaK forms an integral component of the complex. It was shown in DNase I footprinting experiments that refolding and specific binding of HspR to its DNA target does not require DnaK. We conclude that the formation of the stable DnaK-HspR-DNA ternary complex does not depend on the chaperoning activity of DnaK. In affinity chromatography experiments using whole-cell extracts, DnaK was shown to co-purify with HspR, providing additional evidence that the two proteins interact in vivo; it was not possible to purify HspR away from DnaK in any experiments unless a powerful denaturant was used. The level of heat shock induction of chromosomal DnaK could be partially suppressed by expressing dnaK extrachromosomally from a heterologous promoter. In addition, it is shown that DnaK confers enhanced HspR-mediated repression of transcription in vitro. Taken together, these results suggest that DnaK functions as a transcriptional co-repressor by binding to HspR at its operator sites. In this model, the DnaK-HspR system would represent a novel example of feedback regulation of gene expression by a molecular chaperone, in which DnaK directly activates a repressor, rather than inactivates an activator (as is the case in the DnaK-sigma32 and Hsp70-HSF systems of other organisms).

Cyclophilin-promoted folding of mouse dihydrofolate reductase does not include the slow conversion of the late-folding intermediate to the active enzyme.

Cyclophilins accelerate slow protein folding reactions in vitro by catalyzing the cis/trans isomerization of peptidyl-prolyl bonds. Cyclophilins were reported to be involved in a variety of cellular functions, including the promotion of protein folding by use of the substrate mouse dihydrofolate reductase (DHFR). The interaction of cyclophilin with DHFR has only been studied under limited conditions so far, not taking into account that native DHFR exists in equilibrium with a non-native late-folding intermediate. Here we report a systematic analysis of catalysis of DHFR folding by cyclophilins. The specific ligand methotrexate traps DHFR in its native state, permitting a specific analysis of the action of cyclophilin on both denatured DHFR with non-native prolyl bonds and denatured DHFR with all-native prolyl bonds. Cyclophilins from yeast and Neurospora crassa as well as the related prolyl isomerase b from Escherichia coli promote the folding of different forms of DHFR to the enzymatically active form, demonstrating the generality of cyclophilin-catalyzed folding of DHFR. The slow equilibrium between the late-folding intermediate and native DHFR suggests that prolyl isomerization may be required for this final phase of conversion to native DHFR. However, by reversible trapping of the intermediate, we analyze the slow interconversion between native and late-folding conformations in the backward and forward reactions and show a complete independence of cyclophilin. We conclude that cyclophilin catalyzes folding of DHFR, but surprisingly not in the last slow folding step.

Substrate specificity of the SecB chaperone.

The bacterial chaperone SecB assists translocation of proteins across the inner membrane. The mechanism by which it differentiates between secretory and cytosolic proteins is poorly understood. To identify its binding motif, we screened 2688 peptides covering sequences of 23 proteins for SecB binding. The motif is approximately 9 residues long and is enriched in aromatic and basic residues, whereas acidic residues are disfavored. Its identification allows the prediction of binding regions within protein sequences with up to 87% accuracy. SecB-binding regions occur statistically every 20-30 residues. The occurrence and affinity of binding regions are similar in SecB-dependent and -independent secretory proteins and in cytosolic proteins, and SecB lacks specificity toward signal sequences. SecB cannot thus differentiate between secretory and non-secretory proteins via its binding specificity. This conclusion is supported by the finding that SecB binds denatured luciferase, thereby allowing subsequent refolding by the DnaK system. SecB may rather be a general chaperone whose involvement in translocation is mediated by interactions of SecB and signal sequences of SecB-bound preproteins with the translocation apparatus.

D-peptide ligands for the co-chaperone DnaJ.

The molecular chaperone DnaK, the Hsp70 homolog of Escherichia coli, binds hydrophobic polypeptide segments in extended conformation. The co-chaperone DnaJ (Hsp40) has been reported to bind native and denatured proteins as well as peptides. We tested pseudo-peptides of D-amino acids as ligands for both chaperones. In comparison to the parent all-L peptide, these mimetics had either enantiomorphic side chain positions combined with retained main chain direction (normal all-D peptide) or unchanged side chain topology together with reverse direction of the peptide backbone (retro all-D peptide). The peptides were labeled with acrylodan (a), and their binding to DnaK and DnaJ was monitored by the accompanying increase in fluorescence intensity. The parent all-L peptide a-CALLLSAARR bound to both DnaK (Kd = 0.1 microM) and DnaJ (Kd = 9.2 microM). In contrast, the normal all-D and retro all-D peptides did not bind to DnaK; they bound, however, to DnaJ with Kd values of 6.8 microM and 0.9 microM, respectively. The emission spectra of the DnaJ-bound peptides suggests that DnaJ bound both D-peptides with the same main chain direction as L-peptides. Binding of the normal all-D and all-L peptides inhibited the DnaJ-induced stimulation of DnaK ATPase. However, binding of the retro all-D analog to DnaJ did not impair the stimulation, indicating the existence of separate binding sites for peptides and DnaK.

Control of the DnaK chaperone cycle by substoichiometric concentrations of the co-chaperones DnaJ and GrpE.

The polypeptide binding and release cycle of the molecular chaperone DnaK (Hsp70) of Escherichia coli is regulated by the two co-chaperones DnaJ and GrpE. Here, we show that the DnaJ-triggered conversion of DnaK.ATP (T state) to DnaK.ADP.Pi (R state), as monitored by intrinsic protein fluorescence, is monophasic and occurs simultaneously with ATP hydrolysis. This is in contrast with the T-->R conversion in the absence of DnaJ which is biphasic, the first phase occurring simultaneously with the hydrolysis of ATP (Theyssen, H., Schuster, H.-P., Packschies, L., Bukau, B., and Reinstein, J. (1996) J. Mol. Biol. 263, 657-670). Apparently, DnaJ not only stimulates ATP hydrolysis but also couples it with conformational changes of DnaK. In the absence of GrpE, DnaJ forms a tight ternary complex with peptide.DnaK.ADP.Pi (Kd = 0.14 microM). However, by monitoring complex formation between DnaK (1 microM) and a fluorophore-labeled peptide in the presence of ATP (1 mM), DnaJ (1 microM), and varying concentrations of the ADP/ATP exchange factor GrpE (0.1-3 microM), substoichiometric concentrations of GrpE were found to shift the equilibrium from the slowly binding and releasing, high-affinity R state of DnaK completely to the fast binding and releasing, low-affinity T state and thus to prevent the formation of a long lived ternary DnaJ. substrate.DnaK.ADP.Pi complex. Under in vivo conditions with an estimated chaperone ratio of DnaK:DnaJ:GrpE = 10:1:3, both DnaJ and GrpE appear to control the chaperone cycle by transient interactions with DnaK.

Glycerol affects the quantification of aspartate and glutamate in acid-hydrolyzed proteins.

Glycerol is widely used in protein isolation pathways to improve folding and solubility of the proteins of interest. Amino acid composition analysis of protein samples hydrolyzed in the presence of glycerol resulted in underestimation of aspartate and glutamate, when compared to hydrolysis in the absence of glycerol. Quantification of free asparagine, aspartic acid, glutamine and glutamic acid hydrolyzed with hydrochloric acid or methanesulfonic acid in the presence of glycerol resulted in poor recoveries of aspartate and glutamate (between 6 and 66%). Gas chromatography-mass spectrometry analysis of the hydrolyzates revealed, as expected, the presence of esterification products. The esters were formed between the primary and secondary hydroxyl groups of the glycerol and both carboxyl groups of the amino acids. Protein samples intended for compositional analysis should be free of glycerol.

The power stroke of the DnaK/DnaJ/GrpE molecular chaperone system.

The molecular chaperone DnaK, the Hsp70 homolog of Escherichia coli, acts in concert with the co-chaperones DnaJ and GrpE. The aim of this study was to identify the particular phase of the peptide binding-release cycle of the DnaK/DnaJ/GrpE system that is directly responsible for the chaperone effects. By real-time kinetic measurements of changes in the intrinsic fluorescence of DnaK and in the fluorescence of dansyl-labeled peptide ligands, the rates of the following steps in the chaperone cycle were determined: (1) binding of target peptide to fast-binding-and-releasing, low-affinity DnaK ATP; (2) DnaJ-triggered conversion of peptide x DnaK x ATP (T state) to slowly-acting, high-affinity peptide x DnaK x ADP x P(i) (R state); (3) switch from R to T state induced by GrpE-facilitated ADP/ATP exchange; (4) release of peptide. Under conditions approximating those in the cell, the apparent rate constants for the T --> R and R --> T conversion were 0.04 s(-1) and 1.0 s, respectively. The clearly rate-limiting T --> R conversion renders the R state a minor form of DnaK that cannot account for the chaperone effects. Because DnaK in the absence of the co-chaperones is chaperone-ineffective, the T state has also to be excluded. Apparently, the slow, ATP-driven conformational change T --> R is the key step in the DnaK/DnaJ/GrpE chaperone cycle underlying the chaperone effects such as the prevention of protein aggregation, disentangling of polypeptide chains and, in the case of eukaryotic Hsp70 homologs, protein translocation through membranes or uncoating of clathrin-coated vesicles.

Nucleotide-dependent complex formation between the Escherichia coli chaperonins GroEL and GroES studied under equilibrium conditions.

Binding of heptameric GroES to the tetradecameric chaperonin GroEL in the absence or presence of nucleotides was investigated by analytical ultracentrifugation. In the absence of nucleotides, the association constant for the binding of GroES to GroEL, K1, was found to be approximately equal to 3 x 10(5) M(-1). The binding of a second GroES heptamer with only one-fourth the affinity of the first one can be neglected at subequimolecular concentrations relative to GroEL. Under these conditions, mainly an asymmetric "bullet"-shaped complex is formed [see also Schmidt et al. (1994) Science 265, 656-659]. In the presence of ADP or ATP analogues such as ATP-gamma-S or AMP-PNP, the affinity to bind GroES increases by at least 2 orders of magnitude depending on the nucleotide concentration. With increasing GroES:GroEL ratios in the presence of 1 mM ATP analogue, up to two GroES oligomers were bound to one GroEL oligomer, forming the symmetrical "American football"-shaped complex with apparently high affinity for the first GroES ring and considerably lower for the second one. These are the first results that provide an accurate and quantitative description of the equilibrium between asymmetrical and symmetrical complexes at relatively high concentrations of GroEL and GroES that are proposed to exist in vivo. We suggest that the increased affinity of GroEL for GroES plays a role in releasing substrate proteins from the central cavity of GroEL after folding under "non-permissive" conditions.

Sequential action of two hsp70 complexes during protein import into mitochondria.

The mitochondrial chaperone mhsp70 mediates protein transport across the inner membrane and protein folding in the matrix. These two reactions are effected by two different mhsp70 complexes. The ADP conformation of mhsp70 favors formation of a complex on the inner membrane; this 'import complex' contains mhsp70, its membrane anchor Tim44 and the nucleotide exchange factor mGrpE. The ATP conformation of mhsp70 favors formation of a complex in the matrix; this 'folding complex' contains mhsp70, the mitochondrial DnaJ homolog Mdj1 and mGrpE. A precursor protein entering the matrix interacts first with the import complex and then with the folding complex. A chaperone can thus function as part of two different complexes within the same organelle.

Structure and function of the dihydropteroate synthase from Staphylococcus aureus.

The gene encoding the dihydropteroate synthase of staphylococcus aureus has been cloned, sequenced and expressed in Escherichia coli. The protein has been purified for biochemical characterization and X-ray crystallographic studies. The enzyme is a dimer in solution, has a steady state kinetic mechanism that suggests random binding of the two substrates and half-site reactivity. The crystal structure of apo-enzyme and a binary complex with the substrate analogue hydroxymethylpterin pyrophosphate were determined at 2.2 A and 2.4 A resolution, respectively. The enzyme belongs to the group of "TIM-barrel" proteins and crystallizes as a non-crystallographic dimer. Only one molecule of the substrate analogue bound per dimer in the crystal. Sequencing of nine sulfonamide-resistant clinical isolates has shown that as many as 14 residues could be involved in resistance development. The residues are distributed over the surface of the protein, which defies a simple interpretation of their roles in resistance. Nevertheless, the three-dimensional structure of the substrate analogue binary complex could give important insight into the molecular mechanism of this enzyme.

Substrate shuttling between the DnaK and GroEL systems indicates a chaperone network promoting protein folding.

GroEL and DnaK with their cofactors constitute the major chaperone systems promoting protein folding in the Escherichia coli cytosol. The ability of GroEL to bind and promote folding of a substrate released from DnaK led to the proposal that the DnaK and GroEL systems act successively along a protein folding pathway. Here we have investigated the role of both systems in preventing aggregation and assisting refolding of firefly luciferase denatured by guanidinium chloride and heat. We find that DnaK and GroEL compete with each other for binding to non-native luciferase. Addition of ATP and co-operating proteins results in release of luciferase from either chaperone in a non-native conformation. Only a small fraction of luciferase molecules released from GroEL can reach the native state. Instead, the released luciferase must bind repeatedly to the DnaK system, and only then is it able to fold to the native state. Thus, during a folding reaction, DnaK and GroEL do not obligatorily act in succession by promoting earlier and later protein folding steps, respectively. Rather, the two chaperone systems and perhaps others can form a lateral network of co-operating proteins. This chaperone network is proposed to be of particular importance for the assisted refolding of proteins that are unfolded by stress treatment such as heat shock and whose size is too large to allow folding inside the substrate binding cavity of the GroEL ring underneath GroES.

Potassium ions and the molecular-chaperone activity of DnaK.

Potassium ions stabilize the DnaK.ADP complex that forms on incubation of nucleotide-free DnaK with ADP or ATP. Generation of the crystallographically defined Mg2+ cluster [Wilbanks, S.M. & McKay, D.B. (1995) J. Biol. Chem. 270, 2251-2257], in which two K+ and the nucleotide are bound together with Mg2+ in the ATPase site, appears to be essential for the ATP-induced acceleration of binding of peptide ligands, the ATP-induced release of peptide ligands and for the peptide-induced increase in ATPase activity. Thus, K+ is instrumental in signal transmission between the ATPase site and the peptide-binding site.

Regulatory region C of the E. coli heat shock transcription factor, sigma32, constitutes a DnaK binding site and is conserved among eubacteria.

The E. coli heat shock response is regulated at the transcriptional level through stress-dependent controls of the heat shock promoter-specific sigma32 subunit of RNA polymerase. A key aspect of this regulation, the sensing of stress and transmission of this information to sigma32, involves the chaperone system formed by the DnaK, DnaJ and GrpE heat shock proteins. This system mediates stress- dependent controls of levels and activity of sigma32 which rely, at least in part, on direct association of DnaK and DnaJ with sigma32. We identified DnaK binding sites within the sigma32 sequence by probing a cellulose-bound peptide library scanning sigma32. Two sites with high affinity for DnaK, containing the motifs RKLFFNLR and LRNWRIVK, were located centrally and peripherally, respectively, to the region C of sigma32, previously implicated genetically in chaperone-dependent control of sigma32 levels. Cloning and sequencing of rpoH homologs from five Gram-negative proteobacteria revealed that region C, including the DnaK binding motif central to it, is highly conserved among sigma32 homologs but missing in the other sigma factors. We propose that binding of DnaK to region C is central to a conserved regulatory mechanism allowing the sensing of stress by the heat shock gene transcription machinery.

A cycle of binding and release of the DnaK, DnaJ and GrpE chaperones regulates activity of the Escherichia coli heat shock transcription factor sigma32.

The chaperone system formed by DnaK, DnaJ and GrpE mediates stress-dependent negative modulation of the Escherichia coli heat shock response, probably through association with the heat shock promoter-specific sigma32 subunit of RNA polymerase. Interactions of the DnaK system with sigma32 were analysed. DnaJ and DnaK bind free, but not RNA polymerase-bound, sigma32 with dissociation constants of 20 nM and 5 muM respectively. Association and dissociation rates of DnaJ-sigma32 complexes are 5900- and 20-fold higher respectively than those of DnaK-sigma32 complexes in the absence of ATP. ATP destabilizes DnaK-sigma32 interactions. DnaJ, through rapid association with sigma32 and stimulation of hydrolysis of DnaK-bound ATP, mediates efficient binding of DnaK to sigma32 in the presence of ATP, resulting in DnaK-DnaJ-sigma32 complexes containing ADP. GrpE binding to these complexes stimulates nucleotide release and subsequent complex dissociation by ATP. We propose that the principles of this cycle also operate in other chaperone activities of the DnaK system. DnaK and DnaJ cooperatively inhibit sigma32 activity in heat shock gene transcription and GrpE partially reverses this inhibition. These data indicate that reversible inhibition of sigma32 activity through transient association of DnaK and DnaJ is a central regulatory element of the heat shock response.