alpha-crystallin prevents irreversible protein denaturation and acts cooperatively with other heat-shock proteins to renature the stabilized partially denatured protein in an ATP-dependent manner.

alpha-Crystallin, a major lens protein of approximately 800 kDa with subunits of approximately 20 kDa has previously been shown to act as a chaperone protecting other proteins from stress-induced aggregation. Here it is demonstrated that alpha-crystallin can bind to partially denatured enzymes at 42-43 degrees C and prevent their irreversible aggregation, but cannot prevent loss of enzyme activity. However, the alpha-crystallin-bound enzymes regain activity on interaction with other chaperones. The data indicate that the re-activated enzymes are no longer associated with the alpha-crystallin, and ATP is required for re-activation. When inactive luciferase bound to alpha-crystallin was treated with reticulocyte lysate, a rich source of chaperones, up to 60% of the original luciferase activity could be recovered. Somewhat less re-activation was observed when the alpha-crystallin-bound enzyme was treated with heat-shock protein (HSP)70, HSP40, HSP60 and an ATP-generating system. Similar results were also obtained with citrate synthase. The overall results suggest that alpha-crystallin acts to stabilize denaturing proteins so that they can later be re-activated by other chaperones.     

Modulation of the chaperone activities of Hsc70/Hsp40 by Hsp105alpha and Hsp105beta

Hsp105alpha and Hsp105beta are stress proteins found in various mammals including human, mouse, and rat, which belong to the Hsp105/Hsp110 protein family. To elucidate their physiological functions, we examined here the chaperone activity of these stress proteins. Hsp105alpha and Hsp105beta prevented the aggregation of firefly luciferase during thermal denaturation, whereas the thermally denatured luciferase was not reactivated by itself or by rabbit reticulocyte lysate (RRL). On the other hand, Hsp105alpha and Hsp105beta suppressed the reactivation of thermally denatured luciferase by RRL and of chemically denatured luciferase by Hsc70/Hsp40 or RRL. Furthermore, although Hsp105alpha and Hsp105beta did not show ATPase activity, the addition of Hsp105alpha or Hsp105beta to Hsc70/Hsp40 enhanced the amount of hydrolysis of ATP greater than that of the Hsp40-stimulated Hsc70 ATPase activity. These findings suggest that Hsp105alpha and Hsp105beta are not only chaperones that prevent thermal aggregation of proteins, but also regulators of the Hsc70 chaperone system in mammalian cells.     

ATP causes small heat shock proteins to release denatured protein.

Small heat-shock proteins (sHSPs) are a ubiquitous family of low molecular mass (15-30 kDa) stress proteins that have been found in all organisms. Under stress, sHSPs such as alpha-crystallin can act as chaperones binding partially denatured proteins and preventing further denaturation and aggregation. Recently, it has been proposed that the function of sHSPs is to stabilize stress-denatured protein and then act cooperatively with other HSPs to renature the partially denatured protein in an ATP-dependent manner. However, the process by which this occurs is obscure. As no significant phosphorylation of alpha-crystallin was observed during the renaturation, the role of ATP is not clear. It is now shown that ATP at normal physiological concentrations causes sHSPs to change their confirmation and release denatured protein, allowing other molecular chaperones such as HSP70 to renature the protein and renew its biological activity. In the absence of ATP, sHSPs such as alpha-crystallin are more efficient than HSP70 in preventing stress-induced protein aggregation. This work also indicates that in mammalian systems at normal cellular ATP concentrations, sHSPs are not effective chaperones.     

Effective cotranslational folding of firefly luciferase without chaperones of the Hsp70 family

Molecular chaperones of the Hsp70 family (bacterial DnaK, DnaJ, and GrpE) were shown to be strictly required for refolding of firefly luciferase from a denatured state and thus for effective restoration of its activity. At the same time the luciferase was found to be synthesized in an Escherichia coli cell-free translation system in a highly active state in the extract with no chaperone activity. The addition of the chaperones to the extract during translation did not raise the activity of the enzyme. The abrupt arrest of translation by the addition of a translational inhibitor led to immediate cessation of the enzyme activity accumulation, indicating the cotranslational character of luciferase folding. The results presented suggest that the chaperones of the Hsp70 family are not required for effective cotranslational folding of firefly luciferase.     

Folding of the firefly luciferase polypeptide chain with immobilized C-terminus

Refolding of Photinus pyralis firefly luciferase from a denatured state is a slow process; its rate and productivity depend on molecular chaperones of the Hsp70 family. In contrast, cotranslational folding of the enzyme is fast and productive in the absence of chaperones [Svetlov et al., 2006. Protein Sci. 15, 242-247]. During cotranslational folding, the C-termini of polypeptides are bound to massive particles - ribosomes. The question arises whether the immobilization of the polypeptide C-terminus on a massive particle promotes the folding. To test this experimentally, the luciferase with oligohistidine tag at its C-terminus was prepared. This allowed us to immobilize the protein C-terminal segment on chelating Sepharose beads. Here we show that both immobilized and free chains of urea-denatured enzyme refold with the same rate. At the same time, the immobilization of luciferase results in higher refolding yield due to prevention of inter-molecular aggregation. Chaperones of the Hsp70 family promote refolding of both immobilized and free luciferase polypeptides. The results presented here suggest that the high rate of cotranslational folding is not caused by the immobilization of polypeptide C-termini by itself, but is rather due to a favorable start conformation of the growing polypeptide in the peptidyl-transferase center of the ribosome and/or the strongly vectorial character of the folding from N- to C-terminus during polypeptide synthesis.     

Reversible thermal denaturation of immobilized rhodanese

For the first time, the enzyme rhodanese had been refolded after thermal denaturation. This was previously not possible because of the strong tendency for the soluble enzyme to aggregate at temperatures above 37 degrees C. The present work used rhodanese that was covalently coupled to a solid support under conditions that were found to preserve enzyme activity. Rhodanese was immobilized using an N-hydroxymalonimidyl derivative of Sepharose containing a 6-carbon spacer. The number of immobilized competent active sites was measured by using [35S]SO3(2-) to form an active site persulfide that is the obligatory catalytic intermediate. Soluble enzyme was irreversibly inactivated in 10 min at 52 degrees C. The immobilized enzyme regained at least 30% of its original activity even after boiling for 20 min. The immobilized enzyme had a Km and Vmax that were each approximately 3 times higher than the corresponding values for the native enzyme. After preincubation at high temperatures, progress curves for the immobilized enzyme showed induction periods of up to 5 min before attaining apparently linear steady states. The pH dependence of the activity was the same for both the soluble and the immobilized enzyme. These results indicate significant stabilization of rhodanese after immobilization, and instabilities caused by adventitious solution components are not the sole reasons for irreversibility of thermal denaturation seen with the soluble enzyme. The results are consistent with models for rhodanese that invoke protein association as a major cause of inactivation of the enzyme. Furthermore, the induction period in the progress curves is consistent with studies which show that rhodanese refolding proceeds through intermediate states.     

Folding of the firefly luciferase polypeptide chain with the immobilized C terminus

Refolding of firefly Photinus pyralis luciferase from a denatured state is a slow process; its rate and productivity depend on molecular chaperones of the Hsp70 family. In contrast, cotranslational folding of luciferase is fast and productive in the absence of chaperones. During cotranslational folding, the C termini of polypeptides are associated with ribosomes, massive particles. The question arises as to whether C-terminal immobilization on a massive particle promotes folding. To study this problem experimentally, luciferase was C-tagged with hexahistidine to allow its C-terminal immobilization of chelating Sepharose. Both immobilized and free chains of the urea-denatured enzyme refolded at the same rate. At the same time, immobilization led to a higher refolding yield owing to the prevention of intermolecular aggregation. Chaperones of the Hsp70 family promoted folding of both immobilized and free luciferase polypeptides. It was assumed that the high rate of cotranslational folding is not ensured by mere immobilization of the C terminus of the polypeptide, but is rather due to a favorable start conformation of the growing peptide in the peptidyltransferase center of the ribosome and/or the vectorial character of the folding, proceeding from the N to the C end during polypeptide synthesis.     

A small heat shock protein stably binds heat-denatured model substrates and can maintain a substrate in a folding-competent state.

The small heat shock proteins (sHSPs) recently have been reported to have molecular chaperone activity in vitro; however, the mechanism of this activity is poorly defined. We found that HSP18.1, a dodecameric sHSP from pea, prevented the aggregation of malate dehydrogenase (MDH) and glyceraldehyde-3-phosphate dehydrogenase heated to 45 degrees C. Under conditions in which HSP18.1 prevented aggregation of substrates, size-exclusion chromatography and electron microscopy revealed that denatured substrates coated the HSP18.1 dodecamers to form expanded complexes. SDS-PAGE of isolated complexes demonstrated that each HSP18.1 dodecamer can bind the equivalent of 12 MDH monomers, indicating that HSP18.1 has a large capacity for non-native substrates compared with other known molecular chaperones. Photoincorporation of the hydrophobic probe 1,1'-bi(4-anilino)naphthalene-5,5'-disulfonic acid (bis-ANS) into a conserved C-terminal region of HSP18.1 increased reversibly with increasing temperature, but was blocked by prior binding of MDH, suggesting that bis-ANS incorporates proximal to substrate binding regions and that substrate-HSP18.1 interactions are hydrophobic. We also show that heat-denatured firefly luciferase bound to HSP18.1, in contrast to heat-aggregated luciferase, can be reactivated in the presence of rabbit reticulocyte or wheat germ extracts in an ATP-dependent process. These data support a model in which sHSPs prevent protein aggregation and facilitate substrate refolding in conjunction with other molecular chaperones.     

Glucose oxidase immobilization on a novel cellulose acetate-polymethylmethacrylate membrane

Glucose oxidase (GOD) was immobilized on cellulose acetate-polymethylmethacrylate (CA-PMMA) membrane. The immobilized GOD showed better performance as compared to the free enzyme in terms of thermal stability retaining 46% of the original activity at 70 degrees C where the original activity corresponded to that obtained at 20 degrees C. FT-IR and SEM were employed to study the membrane morphology and structure after treatment at 70 degrees C. The pH profile of the immobilized and the free enzyme was found to be similar. A 2.4-fold increase in Km value was observed after immobilization whereas Vmax value was lower for the immobilized GOD. Immobilized glucose oxidase showed improved operational stability by maintaining 33% of the initial activity after 35 cycles of repeated use and was found to retain 94% of activity after 1 month storage period. Improved resistance against urea denaturation was achieved and the immobilized glucose oxidase retained 50% of the activity without urea in the presence of 5M urea whereas free enzyme retained only 8% activity.     

Structural and catalytic properties of L-alanine dehydrogenase from Bacillus cereus

Alanine dehydrogenase from Bacillus cereus, a non-allosteric enzyme composed of six identical subunits, was purified to homogeneity by chromatography on blue-Sepharose and Sepharose 6B-CL. Like other pyridine-linked dehydrogenases, alanine dehydrogenase is inhibited by Cibacron blue, competitively with respect to NADH and noncompetitively with respect to pyruvate. The enzyme was inactivated by 0.1 M glycine/HCl (pH 2) and reactivated by 0.1 M phosphate (pH 8) supplemented with NAD+ or NADH. The reactivation was characterized by sigmoidal kinetics indicating a complex mechanism involving rate-limiting folding and association steps. Cibacron blue interfered with renaturation, presumably by competition with NADH. Chromatography on Sepharose 6B-CL of the partially renatured alanine dehydrogenase led to the separation of several intermediates, but only the hexamer was characterized by enzymatic activity. By immobilization on Sepharose 4B, alanine dehydrogenase from B. cereus retained 66% of the specific activity of the soluble enzyme. After denaturation of immobilized alanine dehydrogenase with 7 M urea, 37% of the initial protein was still bound to Sepharose, indicating that on the average the hexamer was attached to the matrix via, at most, two subunits. The ability of the denatured, immobilized subunits to pick up subunits from solution shows their capacity to fold back to the native conformation after urea treatment. The formation of "hybrids" between subunits of enzyme from B. cereus and Bacillus subtilis demonstrates the close resemblance of the tertiary and quaternary structures of alanine dehydrogenases from these species.     

DnaK, DnaJ and GrpE form a cellular chaperone machinery capable of repairing heat-induced protein damage.

Members of the conserved Hsp70 chaperone family are assumed to constitute a main cellular system for the prevention and the amelioration of stress-induced protein damage, though little direct evidence exists for this function. We investigated the roles of the DnaK (Hsp70), DnaJ and GrpE chaperones of Escherichia coli in prevention and repair of thermally induced protein damage using firefly luciferase as a test substrate. In vivo, luciferase was rapidly inactivated at 42 degrees C, but was efficiently reactivated to 50% of its initial activity during subsequent incubation at 30 degrees C. DnaK, DnaJ and GrpE did not prevent luciferase inactivation, but were essential for its reactivation. In vitro, reactivation of heat-inactivated luciferase to 80% of its initial activity required the combined activity of DnaK, DnaJ and GrpE as well as ATP, but not GroEL and GroES. DnaJ associated with denatured luciferase, targeted DnaK to the substrate and co-operated with DnaK to prevent luciferase aggregation at 42 degrees C, an activity that was required for subsequent reactivation. The protein repair function of DnaK, GrpE and, in particular, DnaJ is likely to be part of the role of these proteins in regulation of the heat shock response.     

Optimization of expression and purification of mitochondrial HSP 40 (Tid1-L) chaperone: Role of mortalin and tid1 in the reactivation and amyloid inhibition of proteins

Stimulation of complex chaperone activity may be a viable means of therapy for neurodegenerative diseases. These chaperons execute reactivation of thermally and chemically aggregated protein substrates by cooperating with their partner co-chaperons. We optimized the expression and purification conditions of Tid1-L chaperone. Expression of Tid1-L in E. coli resulted in the formation of inclusion bodies which was further purified to soluble active form using 8 M urea and Ni-NTA column. Also, we investigated the events of the reactivation and disaggregation using aggregated G6PDH, luciferase and insulin as substrates. Incubation of aggregated/denatured enzymes with mortalin but not with Tid1 and/or Mge1 resulted in the initiation of the disaggregation reaction albeit very insignificantly. Under the same conditions coincubating the samples with chaperon and its assisted partners Tid1-L and nucleotide exchange factor Mge1 led to (40%) increase in enzyme activity of G6PDH. Similarly, luciferase activity was synergistically enhanced in the presence of mortlain/Tid1-L/Mge1 chaperones machinery. Chaperone-dependent disaggregation of thermally aggregated insulin showed that addition of Hsp70 and Hsp40 chaperones resulted in fast-track of renaissance reaction and inhibition of amyloid. The present study results conclude the quality of cell-control involves interaction of chaperon Hsp70 and its co-chaperones leading to complex formation with chemically/thermally aggregated substrate eventually causing its reactivation and disaggregation.     

alpha-B- and alpha-A-crystallin prevent irreversible acidification-induced protein denaturation

alpha-Crystallin (alpha), a major structural protein of the mammalian lens, is a large, physically heterogeneous macromolecule with an average molecular weight of approximately 800 kDa and is composed of two 20-kDa polypeptides designated as alphaA and alphaB. A line of evidence strongly suggests that alphaB may have an essential nonlenticular function. Here it is demonstrated that alphaB can bind partially denatured enzymes effectively at acidic pH and prevent their irreversible aggregation, but cannot prevent loss of enzyme activity. However, when the inactive luciferase bound to alphaB was treated with reticulocyte lysate (a rich source of molecular chaperones) and an ATP-generating system, more than 50% of the original luciferase activity could be recovered. Somewhat less activation was observed when alphaA-bound enzyme or the alpha-bound enzyme was renatured similarly. The overall results suggest that alpha acts as a chaperone to stabilize denaturing proteins at acidic pH so that at a later time they can be reactivated by other chaperones.     

Immobilization of Paenibacillus macerans NRRL B-3186 cyclodextrin glucosyltransferase and properties of the immobilized enzyme

Cyclodextrin glucosyltransferase from Paenibacillus macerans NRRL B-3186 was immobilized on aminated polyvinylchloride (PVC) by covalent binding with a bifunctional agent (glutaraldehyde). The immobilized activity was affected by the length of the hydrocarbon chain attached to the PVC matrix, the amount of the protein loaded on the PVC carrier, and glutaraldehyde concentration. The activity of the immobilized enzyme was 121 units/gram carrier, the specific activity calculated on bound protein basis was 48% of the soluble enzyme. Compared to the free enzyme, the immobilized form exhibited: a higher optimal reaction temperature and energy of activation, a higher K_m (Michaelis constant) and lower V_max (maximal reaction rate), improved thermal stability and resistance to chemical denaturation. The operational stability was evaluated in repeated batch process and the immobilized enzyme retained about 85% of the initial catalytic activity after being used for 14 cycles.     

Comparative study of stability of soluble and cell wall invertase from Saccharomyces cerevisiae

Yeast Saccharomyces cerevisiae is the most significant source of enzyme invertase. It is mainly used in the food industry as a soluble or immobilized enzyme. The greatest amount of invertase is located in the periplasmic space in yeast. In this work, it was isolated into two forms of enzyme from yeast S. cerevisiae cell, soluble and cell wall invertase (CWI). Both forms of enzyme showed same temperature optimum (60°C), similar pH optimum, and kinetic parameters. The significant difference between these biocatalysts was observed in their thermal stability, stability in urea and methanol solution. At 60°C, CWI had 1.7 times longer half-life than soluble enzyme, while at 70°C CWI showed 8.7 times longer half-life than soluble enzyme. After 2-hr of incubation in 8?M urea solution, soluble invertase and CWI retained 10 and 60% of its initial activity, respectively. During 22?hr of incubation of both enzymes in 30 and 40% methanol, soluble invertase was completely inactivated, while CWI changed its activity within the experimental error. Therefore, soluble invertase and CWI have not shown any substantial difference, but CWI showed better thermal stability and stability in some of the typical protein-denaturing agents.     

Comparison of Hsc70 orthologs from polar and temperate notothenioid fishes: differences in prevention of aggregation and refolding of denatured proteins

Although a great deal is known about the cellular function of molecular chaperones in general, very little is known about the effect of temperature selection on the function of molecular chaperones in nonmodel organisms. One major unanswered question is whether orthologous variants of a molecular chaperone from differential thermally adapted species vary in their thermal responses. To address this issue, we utilized a comparative approach to examine the temperature interactions of a major cytosolic molecular chaperone, Hsc70, from differently thermally adapted notothenioids. Using in vitro assays, we measured the ability of Hsc70 to prevent thermal aggregation of lactate dehydrogenase (LDH). We further compared the capacity of Hsc70 to refold chemically denatured LDH over the temperature range of -2 to +45 degrees C. Hsc70 purified from the temperate species exhibited greater ability to prevent the thermal denaturation of LDH at 55 degrees C compared with Hsc70 from the cold-adapted species. Furthermore, Hsc70 from the Antarctic species lost the ability to competently refold chemically denatured LDH at a lower temperature compared with Hsc70 from the temperate species. These data indicate the function of Hsc70 in notothenioid fishes maps onto their thermal history and that temperature selection has acted on these molecular chaperones.     

The molecular chaperone Hsc70 from a eurythermal marine goby exhibits temperature insensitivity during luciferase refolding assays

The role and function of molecular chaperones has been widely studied in model systems (e.g. yeast, Escherichia coli and cultured mammalian cells), however, comparatively little is known about the function of molecular chaperones in eurythermal ectotherms. To investigate the thermal sensitivity of molecular chaperone function in non-model ectotherms, we examined the in vitro activity of Hsc70, a constitutively expressed member of the 70-kDa heat-shock protein gene family, purified from white muscle of the eurythermal marine goby Gillichthys mirabilis. The activity of G. mirabilis Hsc70 was assessed with an in vitro refolding assay where the percent refolding of thermally denatured luciferase was monitored using a luminometer. Assays were conducted from 10-40 degrees C, a range of temperatures that is ecologically relevant for this estuarine species. The results showed that isolated Hsc70 displayed chaperone characteristics in vitro, and was relatively thermally insensitive across the range of experimental temperatures. In addition, the thermal stability of the luciferase refolding capacity of Hsc70 was relatively stable, with refolding activity occurring as high as 50 degrees C. Overall, Hsc70 from G. mirabilis displayed thermal properties in vitro that suggest that the molecular chaperone is capable of binding and chaperoning proteins at temperatures that the goby encounters in nature.     

Refolding of firefly luciferase immobilized on agarose beads

The renaturation yield of the denatured firefly luciferase decreased strongly with increasing protein concentration in a renaturation buffer, because of aggregation. In this study, firefly luciferase was immobilized on agarose beads at a high concentration. Although the protein concentration was extremely high (about 100-fold) compared to that of soluble luciferase, the renaturation yield was comparable with that for the soluble one. Thus, immobilization was shown to be effective for avoiding aggregation of firefly luciferase. It was also shown that the optimum buffer conditions for renaturation of the immobilized luciferase were the same as those for the renaturation in solution. Also, it was indicated that electrostatic interactions between a protein and the matrix have a negative effect on renaturation of the immobilized luciferase since the renaturation yield decreased at acidic pH only for the immobilized luciferase. These novel observations are described in detail in this paper.     

α-Chymotrypsin stability in aqueous-acetonitrile mixtures: is the native enzyme thermodynamically or kinetically stable under low water conditions?

Like many proteins, α-chymotrypsin is denatured in 50% volume aqueous-acetonitrile mixtures. However, it also shows high catalytic activity in 70% or more acetonitrile. Good activity in two different aqueous organic composition ranges has been described for several other enzymes. The stability of the native protein under low water conditions is generally believed to be a kinetic phenomenon, though there are also arguments for thermodynamic stability. We have distinguished between these possibilities by studying the effects of changing medium composition at different times. In preliminary experiments, we found catalytic activity could be recovered by adding neat acetonitrile to chymotrypsin in a 50% mixture, suggesting that the enzyme could renature under these conditions. However, in the 50% mixture, the true initial activity at 30°C is not zero, as the literature suggests. Instead, there is an initial burst of product formation over a few minutes, after which the enzyme becomes inactivated. By pre-incubating a 50% aqueous-acetonitrile mixture at 30°C prior to enzyme addition, the product burst could be eliminated. Activity could not then be recovered by slow addition of acetonitrile to the denatured enzyme. In contrast, it was possible to renature by dilution with aqueous buffer so that regeneration of catalytic activity was achieved. Thus, the good practical performance at high acetonitrile concentrations almost certainly results from a high kinetic barrier towards denaturation. The kinetics of enzyme denaturation in 50% and 70% acetonitrile were also investigated both at 30 and 20°C. Loss of catalytic activity was faster at higher temperature and at lower acetonitrile concentrations.     

Identification of small molecules that modify the protein folding activity of heat shock protein 70

Molecular chaperones, such as heat shock protein 70 (Hsp70) and its bacterial ortholog DnaK, play numerous important roles in protein folding. In vitro, this activity can be observed by incubating purified chaperones with denatured substrates and measuring the recovery of properly folded protein. In an effort to rapidly identify small molecules that modify this folding activity, we modified an existing method for use in 96-well plates. In this assay, denatured firefly luciferase was treated with a mixture of DnaK and prospective chemical modulators. The luminescence of refolded luciferase was used to follow the reaction progress, and counterscreens excluded compounds that target luciferase; thus, hits from these screens modify protein folding via their effects on the function of the chaperone machine. Using this platform, we screened a pilot chemical library and found five new inhibitors of DnaK and one compound that promoted folding. These chemical probes may be useful in studies aimed at understanding the many varied roles of chaperones in cellular protein folding. Moreover, this assay provides the opportunity to rapidly screen for additional compounds that might regulate the folding activity of Hsp70.