Interaction of hsp70 with unfolded proteins: effects of temperature and nucleotides on the kinetics of binding.

Abstract

Circular dichroism and HPLC gel filtration were used to show that cytosolic hsp7O is thermally stable but undergoes a conformational transition (midpoint, 430C; 57C in the presence of ATP or ADP) leading to oligomerization. hsp7O binds to unfolded, but not to folded, proteins in a temperature-dependent manner; complex formation is significant only at physiologically relevant temperatures. hsp7O binds ADP more tightly than ATP to form a binary complex, which binds to the unfolded protein more rapidly than free hsp7O. ADP also inhibits the ATP-induced dissociation of the hsp7O-protein complex. A regulatory role for the hsp7O-nucleotide binary complexes is proposed.Recent studies indicate that protein folding and assembly events in vivo are mediated by intracellular components now being referred to as molecular chaperones (reviewed in refs. 1-3). One class of these chaperones is the 70-kDa heat shock protein, hsp70. The cytosolic forms of mammalian hsp70 are present in cells as two different gene products (4): a stressinducible form, hsp72, and a constitutive member, hsp73, also known as hsc70. hsp70 family members have been shown to interact with a number of proteins undergoing maturation in the cell (5-8). In addition to these nascent events, it has been proposed that hsp70 proteins can recognize and bind to mature polypeptides that may have become unfolded in the cell (e.g., following heat shock) and thereby facilitate their refolding (9). To date relatively little experimental evidence has been provided to support this idea. There are ample data demonstrating that nascent polypeptides, unable to properly fold or assemble, remain bound to their particular hsp70 chaperone (5, 10, 11). In vitro studies have shown that hsp70 proteins interact with a variety of short synthetic polypeptides (12).In the present study, we have utilized an in vitro system to examine the possible interaction of the mammalian hsp70 with both folded and unfolded polypeptides. We show that the cytosolic hsp73 will form stable complexes with a variety of unfolded protein targets, but not with their properly folded counterparts. Furthermore, we present data regarding the role of temperature and nucleotides in these interactions. MATERIALS AND METHODSMaterials. Bovine brain hsp73 and human hsp72/73 from HeLa cells were isolated and purified as described (13). Reduced carboxymethylated a-lactalbumin (RCMLA), bovine a-lactalbumin (type I) (a-LA), and horse heart cytochrome c (type VI) were obtained from Sigma. Reduced carboxamidomethylated ribonuclease A was prepared as described (14). Staphyloccocal nuclease (SNase) was purified from a cloned system provided by D. (15). Gel-filtration chromatography was performed with a Beckman HPLC instrument using a Bio SEC-250 silica column (600 x 7.5 mm;Bio-Rad) at 22°C; 20 mM sodium phosphate buffer/0.20 M KCl, pH 6.5 (buffer A), was used as the mobile phase; the flow rate was 1 ml/min. Typical elution volumes for hsp73, dimer, trimer, hsp73 soluble aggregates, reduced carboxymethylated a-lactalbu...

A 70-kDa molecular chaperone, DnaK, from the industrial bacterium Bacillus licheniformis: gene cloning, purification and molecular characterization of the recombinant protein

“…The thermally induced unfolding of BlDnaK was completely irreversible as previously showed for bovine and human Hsc70s [51][52][53], but this is not the case for EcDnaK which is capable to recover about 90% of its CD signal at 222 nm after being heated up to 90°C [54]. Differential scanning calorimetry of EcDnaK in the absence of nucleotides shows three unfolding transitions with T m s of 42, 55, and 72°C, respectively [55].…”

Cysteine‐capped gold nanoparticles suppress aggregation of proteins exposed to heat stress

“…BSA is known to be sensitive to conformational changes when it interacts with other molecules, and because of this it is useful in studies involving interaction of protein and nanoparticles (17). hHsp70 is a molecular chaperone that is ubiquitous in the cell and its expression is upregulated by physiological stress (10). We also investigated the capability of AuNPs to suppress the heat-induced aggregation of two aggregation prone proteins, MDH and CS.…”

“…Could AuNPs have protected both hHsp70 and MDH from aggregating in response to thermal stress? Hsp70 proteins are generally heat stable, and in fact, one of their roles is to prevent aggregation of other proteins in the event of physiological stress (10,11). For this reason, it is unlikely that AuNPs acted by stabilizing hHsp70 to enhance its chaperone activity as Hsp70 is inherently a stable protein (10).…”

“…Heat shock protein 70 (Hsp70) is one of the most abundant members of this family of proteins. It is involved in the folding of other proteins (chaperone function) and operates in an ATP-dependent fashion (10). Hsp70 is capable of suppressing the heat-induced aggregation of model proteins such as malate dehydrogenase (MDH) in vitro (11).…”

Firefly luciferase mutants as sensors of proteome stress

“…The mechanistic elucidation of the reaction cycle of Hsp70 came from both in vitro and in vivo studies, primarily using DnaK which has served as a paradigm for all canonical Hsp70s (Figure 4b). Hsp70 has been shown to bind only to unfolded, but not to folded or native proteins in a temperature-dependent manner and the complex of Hsp70 with the nucleotide (ATP or ADP) modulates its intrinsic affinity for the polypeptide (Palleros et al, 1991;Pellecchia et al, 2000). In the ATP bound state, Hsp70 rapidly binds to its polypeptide in an open state where the latch over the peptide binding cleft is open.…”