The structure of ClpB: a molecular chaperone that rescues proteins from an aggregated state

Molecular chaperones assist protein folding by facilitating their "forward" folding and preventing aggregation. However, once aggregates have formed, these chaperones cannot facilitate protein disaggregation. Bacterial ClpB and its eukaryotic homolog Hsp104 are essential proteins of the heat-shock response, which have the remarkable capacity to rescue stress-damaged proteins from an aggregated state. We have determined the structure of Thermus thermophilus ClpB (TClpB) using a combination of X-ray crystallography and cryo-electron microscopy (cryo-EM). Our single-particle reconstruction shows that TClpB forms a two-tiered hexameric ring. The ClpB/Hsp104-linker consists of an 85 A long and mobile coiled coil that is located on the outside of the hexamer. Our mutagenesis and biochemical data show that both the relative position and motion of this coiled coil are critical for chaperone function. Taken together, we propose a mechanism by which an ATP-driven conformational change is coupled to a large coiled-coil motion, which is indispensable for protein disaggregation.     

Heat-resistance and heat-shock response in the nosocomial pathogen Enterococcus faecium

We have characterized the heat-shock response of the nosocomial pathogen Enterococcus faecium. The growth of E. faecium cells was analyzed at different temperatures; little growth was observed at 50 degrees C, and no growth at 52 degrees C or 55 degrees C. In agreement, a marked decrease of general protein synthesis was observed at 52 degrees C, and very light synthesis was detected at 55 degrees C. The heat resistance of E. faecium cells was analyzed by measuring the survival at temperatures higher than 52 degrees C and, after 2 h of incubation, viable cells were still observed at 70 degrees C. By Western blot analysis, two heat-induced proteins were identified as GroEL (65 kDa) and DnaK (75 kDa). Only one isoform for either GroEL or DnaK was found. The gene expression of these heat-shock proteins was also analyzed by pulsed-labeled experiments. The heat-induced proteins showed an increased rate of synthesis during the first 5 min, reaching the highest level of induction after 10 min and returning to the steady-state level after 20 min of heat treatment.     

Insulin binding and degradation studies on erythrocytes at different temperatures

Insulin binding of human erythrocytes has been investigated between 0 and 37 degrees C using porcine 125I-insulin/unlabeled porcine insulin and mono [125I] (Tyr-A14)biosynthetic human insulin/ unlabeled biosynthetic human insulin, respectively. Either system exhibited a regular thermodynamical behavior between 0 and 22 degrees C, giving unitary free-energy changes of about -58/ -59 kJ/mol, unitary entropy changes of about +55/ +70 J/K per mol and a reaction heat of -43.1/ -38.3 kJ/mol. From 22 up to 37 degrees C an irregular thermodynamical behavior could be observed, which can be partially explained by an increased insulin degradation during incubation and an additional time-dependent binding of the degradation products.     

Effect of immobilization on stability and kinetic properties of alpha-L-fucosidase from Turbo cornutus

An amount of alpha-L-fucosidase from T. cornutus liver was copolymerized with glutaraldehyde using bovine serum albumin as the carrier protein. The properties of the native, the soluble enzyme polymer complex, and the insoluble enzyme polymer complex were studied and compared under various conditions of pH, temperature, substrate, and inhibitor concentration. Native alpha-L-fucosidase was heat labile and lost more than 85% of its activity when incubated at 55 degrees C for 5 min. In contrast, under equivalent incubation conditions, both the soluble and the insoluble enzyme polymer complexes exhibited enhanced resistance to thermal inactivation and after 5 min lost only 65 and 40% of their original activity, respectively. Polymerzation also resulted in the shift of pH optima towards the acidic range, a decrease in activation energy and a change in the apparent K(m) values towards the p-nitrophenyl-alpha-L-fucopyranoside substrate.     

Aggregation of human red blood cells after moderate heat treatment

The aggregation behaviour of normal and heat treated (48.4 degrees C, 48.8 degrees C, 49.5 degrees C) red blood cells (RBCs) suspended in dextran-saline solutions (Dx 70, Dx 173) was investigated by a laser light reflectometric method over a wide range of bridging energies. The characteristic times of rouleau formation were found to be increased after RBC heat treatment. The disaggregation shear stress is not significantly different between normal RBCs and heat treated RBCs. The loss of cell deformability is nevertheless shown to improve slightly the dissociation efficiency of the flowing liquid in a shear flow resulting in a small reduction of the disaggregation shear rate after heat treatment. Heat treatment is also shown to alter the structure of RBC network at equilibrium. These results indicate that heat induced alterations of erythrocytes only affects the mechanical properties of the cell membrane without significant changes in the macromolecular bridging energy.     

Thermotolerance and the heat-shock response in Candida albicans

At elevated temperatures, yeast cells of Candida albicans synthesized nine heat-shock proteins (HSPs) with apparent molecular masses of 98, 85, 81, 76, 72, 54, 34, 26 and 18 kDa. The optimum temperature for the heat-shock response was 45 degrees C although HSPs were detected throughout the range 41-46 degrees C. Protein synthesis was not observed in cells kept at 48 degrees C. Yeast cells survived exposure to an otherwise lethal temperature of 55 degrees C when they had previously been exposed to 45 degrees C. The thermotolerance induced during incubation at 45 degrees C required protein synthesis, since protection was markedly reduced by trichodermin. Mercury ions induced a set of three stress proteins, one of which corresponded in size to an HSP, and cadmium ions evoked one stress protein seemingly unrelated to the HSPs observed after temperature shift.     

The occurrence of thermotolerance in non-proliferating rat hepatocytes in primary culture

The occurrence of thermotolerance, induced by an initial heat treatment at 42 degrees C for 30 min, was studied in adult non-proliferating rat hepatocytes in primary culture. Heat treatment at 42 degrees C for 30 min did not affect cell morphology, cell attachment, Na+, K+ pump activity, K+ content and lactate dehydrogenase accumulation into the medium. In contrast, after exposure to 44 degrees C for 30 min a dramatic change in all these parameters was observed. However, of the cells, which remained attached to the substratum 24 h after treatment, Na+, K+ pump activity and K+ content appeared to be normal compared with untreated cells. Cells, pre-treated at 42 degrees C for 30 min, followed by incubation at 37 degrees C for 16 h, were found to be completely thermal resistant against heat treatment at 44 degrees C, as judged by cell morphology, detachment from the substratum, lactate dehydrogenase accumulation, Na+, K+ pump activity and K+ content. These results show that induction and development of thermotolerance can be studied in non-proliferating cells in primary culture.     

Medium tumor antigen of polyomavirus transformation-defective mutant NG59 is associated with 73-kilodalton heat shock protein.

Affinity-purified medium T antigen encoded by NG59, a nontransforming mutant of polyomavirus, is specifically associated with a protein of 72,000 daltons (72K protein). Medium T antigens of wild-type polyomavirus and the transformation-competent mutant dl8 are not associated with the 72K protein. Instead, they form a complex with another protein of 61,000 daltons. Several lines of evidence suggest that the medium T antigen-associated 72K protein is equivalent to the abundant and constitutive 73K heat shock protein. First, on two-dimensional polyacrylamide gels the 72K protein migrated with the same pI (5.6) as did the 73K heat shock protein. Second, the 72K protein was immunoprecipitable with antibodies against heat shock proteins. Third, when digested with V8 protease, the 72K protein gave rise to the same pattern of fragments as did the 73K heat shock protein.     

Thermoprotection of preimplantation bovine embryos from heat shock by glutathione and taurine.

To determine if deleterious effects of heat shock on embryos could be reduced in vitro by glutathione or taurine, morulae from superovulated cows were placed in modified Hams-F10 medium supplemented with 50 nM glutathione (GSH), 50 mM taurine or neither. Morulae were incubated for 2 hours at 38.5 degrees C, then at 42.0 degrees C (heat shock) or 38.5 degrees C for 2 hours and followed by incubation at 38.5 degrees C for 20 hours. Neither GSH nor taurine enhanced viability or blastocyst development at 38.5 degrees C. At 42.0 degrees C, however, GSH and taurine increased (P less than 0.02) viability (73%, 41% and 26% live for GSH, taurine and control); GSH increased (P less than 0.05) blastocyst development (55% for GSH vs. 30% for control). In conclusion, partial thermoprotection of bovine embryos from heat shock can be achieved in vitro by administration of GSH. Taurine is only slightly effective.     

Heat shock-induced translational alterations in HeLa cells. Initiation factor modifications and the inhibition of translation

Heat shock at 45 degrees C virtually abolishes protein synthesis in HeLa cells, but return to 37 degrees C effects a complete recovery and the concomitant synthesis of heat shock-induced proteins. Heat shock induces polysome disaggregation, indicating initiation is principally inhibited. In vitro assays for initiation factor activities reveal heat shock inhibits eukaryotic initiation factor 2 (eIF-2), eIF-(3 + 4F), and eIF-4B. Immunoblot analyses show that eIF-2 alpha and eIF-2 beta become modified during heat shock, and eIF-4B variants disappear. Upon return to 37 degrees C, these alterations reverse. The modifications of eIF-2 alpha and eIF-4B are due to phosphorylation and dephosphorylation, respectively. Enzymatic activities induced by heat shock inhibit protein synthesis and modify initiation factors in a rabbit reticulocyte lysate. Initiation factor modifications may contribute to, or cause, protein synthesis inhibition.     

Thermal adaptation in CHO cells at 40 degrees C: the influence of growth conditions and the role of heat shock proteins

The kinetics of thermal adaptation at the nonlethal temperature of 40 degrees C was studied in CHO (Chinese hamster ovary) cells in vitro. Thermal resistance, demonstrated as an increase in mean 45 degrees C killing time or as an increase in the shoulder of the 45 degrees C survival curve, was fully developed by 2 h. Control cells in early logarithmic phase were more heat sensitive than those in stationary phase. Corresponding 45 degrees C killing time frequency distributions were unimodal with an increase in mean killing time from early logarithmic to stationary phase. Cells which were thermally adapted at 40 degrees C for 6 h had biphasic 45 degrees C killing time frequency distributions, and as cells progressed from early logarithmic to stationary phase the heat-sensitive subpopulation progressively declined. Exposure to 40 degrees C produced a 30% increase in total protein synthesis. Proteins with molecular weights 72, 89, and 109 kDa which correspond to those induced by lethal heat shock were synthesized at 40 degrees C, but there was no close temporal correlation between the development of heat resistance at 40 degrees C and synthesis of the heat shock proteins. Cycloheximide (100 micrograms/ml) reduced the mean 45 degrees C killing time but did not totally prevent the development of heat resistance at 40 degrees C.     

Structural studies of the BstVI restriction-modification proteins by fluorescence spectroscopy.

Structural studies of the proteins of the BstVI restriction-modification system of Bacillus stearothermophilus V were carried out using intrinsic fluorescence techniques. The exposure and environments of their tryptophanyl residues were determined using collisional quenchers. Quenching of BstVI endonuclease by iodide suggested a heterogeneous class of tryptophan residues, while the results obtained with M.BstVI methylase were consistent with a rather exposed tryptophan population. A comparison of the quenching efficiencies at 20 degrees C and 55 or 60 degrees C showed that their structures are more flexible and open at the temperature at which they exhibit maximal activity. The endonuclease reached its active conformation only after 1 h of incubation at 60 degrees C. Fluorescence changes were observed upon Mn2+ and Mg2+ binding, with Kd values in the range 3-5 microM. The binding of S-adenosyl-L-methionine to the methylase produced conformational changes, which were consistent with binding to a single site of Kd 550 and 680 microM at 20 degrees C and 55 degrees C, respectively. Quenching experiments with iodide showed that the presence of S-adenosyl-L-methionine leads to different conformational states at 20 degrees C and 55 degrees C. These results were interpreted in terms of differences in the structural characteristics of these restriction-modification proteins as well as in terms of differences in the conformational states that these enzymes exhibit at 20 degrees C and at the temperature at which they are most active.     

Properties of Na+, K+-ATPase of liver plasma membranes in the hamster

This study was designed to establish the properties of liver plasma membranes (LPM) Na+,K+-ATPase in the hamster and to determine whether a similar assay may be used to measure enzyme activity in the hamster and in the rat. Maximal Na+,K+-ATPase activity was obtained when the assay medium contained 5 mM Mg APT2- with or without 1 mM free Mg2+, 120 mM Na+, 12,5 mM K+. The incubation must be performed at 37 degrees C, pH 7.4. In the absence of free Mg2+, the saturation curve with respect to the substrate Mg ATP2- resulted in biphasic complex kinetics with a maximal activity at a substrate concentration of 5 mM. In the presence of 1 mM free Mg2+ activation of Na+,K+-ATPase and modification of the kinetics were observed: the biphasic curve tended to disappear and to become of the Michaelis-Menten type. The apparent Km for Mg APT2- was 0.36 mM and the Vmax 34.5 mumol.h-1.mg protein-1. In the presence of 10 mM free Mg2+ a decrease in the Vmax was observed without any effect on the apparent Km for Mg APT2-. It is concluded that the same incubation medium may be used to assay LPM N+,K+-ATPase from hamster and rat and that the addition of 1 mM free Mg2+ to the incubation medium is recommended to obtain Michaelis-Menten kinetics in order to eliminate complex kinetics due to the absence of free Mg2+.     

Aggregation and disaggregation kinetics of human blood platelets: Part II. Shear-induced platelet aggregation.

A population balance equation (PBE) mathematical model for analyzing platelet aggregation kinetics was developed in Part I (Huang, P. Y., and J. D. Hellums. 1993. Biophys. J. 65: 334-343) of a set of three papers. In this paper, Part II, platelet aggregation and related reactions are studied in the uniform, known shear stress field of a rotational viscometer, and interpreted by means of the model. Experimental determinations are made of the platelet-aggregate particle size distributions as they evolve in time under the aggregating influence of shear stress. The PBE model is shown to give good agreement with experimental determinations when either a reversible (aggregation and disaggregation) or an irreversible (no disaggregation) form of the model is used. This finding suggests that for the experimental conditions studied disaggregation processes are of only secondary importance. During shear-induced platelet aggregation, only a small fraction of platelet collisions result in the binding together of the involved platelets. The modified collision efficiency is approximately zero for shear rates below 3000 s-1. It increases with shear rates above 3000 s-1 to about 0.01 for a shear rate of 8000 s-1. Addition of platelet chemical agonists yields order of magnitude increases in collision efficiency. The collision efficiency for shear-induced platelet aggregation is about an order of magnitude less at 37 degrees C than at 24 degrees C. The PBE model gives a much more accurate representation of aggregation kinetics than an earlier model based on a monodispersed particle size distribution.     

Acquired thermotolerance and heat shock proteins in thermophiles from the three phylogenetic domains.

Thermophilic organisms from each of the three phylogenetic domains (Bacteria, Archaea, and Eucarya) acquired thermotolerance after heat shock. Bacillus caldolyticus grown at 60 degrees C and heat shocked at 69 degrees C for 10 min showed thermotolerance at 74 degrees C, Sulfolobus shibatae grown at 70 degrees C and heat shocked at 88 degrees C for 60 min showed thermotolerance at 95 degrees C, and Thermomyces lanuginosus grown at 50 degrees C and heat shocked at 55 degrees C for 60 min showed thermotolerance at 58 degrees C. Determinations of protein synthesis during heat shock revealed differences in the dominant heat shock proteins for each species. For B. caldolyticus, a 70-kDa protein dominated while for S. shibatae, a 55-kDa protein dominated and for T. lanuginosus, 31- to 33-kDa proteins dominated. Reagents that disrupted normal protein synthesis during heat shock prevented the enhanced thermotolerance.     

Enhancement of the thermostability of thermophilic bacterium PS-3 PPase on substitution of Ser-89 with carboxylic amino acids

Serine 89 of the inorganic pyrophosphatase (PPase) subunit from thermophilic bacterium PS-3 (PS-3) was replaced with glycine, alanine, threonine, glutamic acid, or aspartic acid by the PCR-mutagenesis method with Mut-1 in order to determine the contribution of this serine residue to the thermostability and structural integrity of the enzyme molecule. S89G, S89A, and S89T showed reduced catalytic activity, whereas S89D and S89E showed increased enzyme activity. S89G, S89A, and S89T as well as the wild-type PPase were stable in the presence of 5 mM MgCl(2) at 70 degrees C for 1 h, but were inactivated rapidly with time at 80 degrees C. On the contrary, S89D and S89E were stable at 80 degrees C, showing more than 95% of the original activity after 1 h incubation. The wild-type PPase, S89D and S89E were each a hexamer before and after incubation at 80 degrees C for 1 h, while S89G and S89A comprised a mixture of a hexamer and a trimer both before and after incubation at 80 degrees C for 1 h. On the other hand, S89T was a mixture of a hexamer, a trimer and a monomer, and it was partially precipitated during heat treatment at 80 degrees C. The CD spectra of the recombinant enzymes in the far-ultraviolet region were the same as that of the wild-type PPase, whereas those of S89G, S89A, and S89T as well as the wild-type PPase were markedly different after heat treatment, although those of S89D and S89E did not change. The present study suggested that local small change(s) in the network of interactions among amino acid residues on replacement at position 89 led to the PS-3 PPase molecule being unable to form a hexamer from trimers or to dissociate into monomers in some cases without a significant change in the backbone conformation. It was also suggested that the partial disordering of the conformation of PS-3 PPase caused by heat depended on the degree of hydrophilicity in the vicinity of position 89.     

Heat shock protein synthesis and thermotolerance in Salmonella typhimurium

The resistance of stationary phase Salmonella typhimurium to heating at 55 degrees C was greater in cells grown in nutritionally rich than in minimal media, but in all media tested resistance was enhanced by exposing cells to a primary heat shock at 48 degrees C. Chloramphenicol reduced the acquisition of thermotolerance in all media but did not completely prevent it in any. The onset of thermotolerance was accompanied by increased synthesis of major heat shock proteins of molecular weight about 83, 72, 64 and 25 kDa. When cells were shifted from 48 degrees C to 37 degrees C, however, thermotolerance was rapidly lost with no corresponding decrease in the levels of these proteins. There is thus no direct relationship between thermotolerance and the cellular content of the major heat shock proteins. One minor protein of molecular weight about 34 kDa disappeared rapidly following a temperature down-shift. Its presence in the cell was thus correlated with the thermotolerant state.     

Interaction of the fluorescent dye 1-N-phenylnaphthylamine with Escherichia coli cells during heat stress and recovery from heat stress

The fluorescent dye 1-N-phenylnaphthylamine permeated Escherichia coli cells after exposure to a heat stress at 55 degrees C in Tris/Mg2+ buffer, pH 8.0. The rate of dye permeation increased with time during heat treatment and decreased gradually during subsequent incubation at 37 degrees C in a minimal medium. The initial level of rapid adsorption of the dye also increased with heating time, although it remained roughly constant during post-heating incubation. The results obtained suggest that the permeability barrier to the dye in the outer membrane was damaged by heat stress and was repaired after sublethal heating. RNA, protein and lipid syntheses, as well as an energy-yielding process, appeared to be necessary for the repair of impermeability to the dye.