Interactions of bovine serum albumin with ethylene oxide/butylene oxide copolymers in aqueous solution

The interactions of bovine serum albumin (BSA) with three ethylene oxide/butylene oxide (E/B) copolymers having different block lengths and varying molecular architectures is examined in this study in aqueous solutions. Dynamic light scattering (DLS) indicates the absence of BSA-polymer binding in micellar systems of copolymers with lengthy hydrophilic blocks. On the contrary, stable protein-polymer aggregates were observed in the case of E 18B 10 block copolymer. Results from DLS and SAXS suggest the dissociation of E/B copolymer micelles in the presence of protein and the absorption of polymer chains to BSA surface. At high protein loadings, bound BSA adopts a more compact conformation in solution. The secondary structure of the protein remains essentially unaffected even at high polymer concentrations. Raman spectroscopy was used to give insight to the configurations of the bound molecules in concentrated solutions. In the vicinity of the critical gel concentration of E 18B 10 introduction of BSA can dramatically modify the phase diagram, inducing a gel-sol-gel transition. The overall picture of the interaction diagram of the E 18B 10-BSA reflects the shrinkage of the suspended particles due to destabilization of micelles induced by BSA and the gelator nature of the globular protein. SAXS and rheology were used to further characterize the structure and flow behavior of the polymer-protein hybrid gels and sols.     

Effect of denaturants on multisite and unisite ATP hydrolysis by bovine heart submitochondrial particles with and without inhibitor protein

The effect of guanidinium hydrochloride (GdnHCl) on multisite and unisite ATPase activity by F0F1 of submitochondrial particles from bovine hearts was studied. In particles without control by the inhibitor protein, 50 mM GdnHCl inhibited multisite hydrolysis by about 85%; full inhibition required around 500 mM. In the range of 500-650 mM, GdnHCl enhanced the rate of unisite catalysis by promoting product release; it also increased the rate of hydrolysis of ATP bound to the catalytic site without GdnHCl. GdnHCl diminished the affinity of the enzyme for aurovertin. The effects of GdnHCl were irreversible. The results suggest that disruption of intersubunit contacts in F0F1 abolishes multisite hydrolysis and stimulates of unisite hydrolysis. Particles under control by the inhibitor protein were insensitive to concentrations of GdnHCl that induce the aforementioned alterations of F0F1 free of inhibitor protein, indicating that the protein stabilizes the global structure of particulate F1.     

Intermolecular interactions in solutions of serum albumin

The mechanisms of intermolecular protein complex formation were studied by the example of monomers, oligomers and aggregates of bovine serum albumin (BSA) depending on the protein concentration, pH and urea concentration. Using dynamic light scattering (DLS), analytical ultracentrifugation (AUC) and PAG electrophoresis we have shown the existence of dynamic equilibrium between monomers and aggregates in BSA solution. Decreasing pH of the solution (4.0–1.0) resulted in increasing sizes of the aggregates. In the solutions with low urea concentrations (below 2 M) the sizes of aggregates decreased, while higher urea concentrations (2–8 M) induced formation of larger aggregates due to the unfolding of the protein.     

Sphingomyelinase-to-LDL molar ratio determines low density lipoprotein aggregation size: biological significance

The subendothelial retention of low density lipoproteins (LDL) is believed to be the central pathogenic event in atherosclerosis, as stated by the response-to-retention hypothesis. Sphingomyelinase, an enzyme present in the arteries, has been proven to promote LDL aggregation. This study investigates the hypothesis that the extent of LDL aggregation is determined by the molar ratio of sphingomyelinase (SMase)-to-LDL, rather than the absolute concentrations. A mass action model is used to describe the aggregation process, and binding and dissociation rate constants are determined by fitting of dynamic light scattering data. The model predicts aggregate sizes that agree well with experimental observations. This study also tests the hypothesis that monocyte uptake of LDL correlates with aggregate size. LDL aggregates of three specific sizes (75, 100, and 150 nm) were incubated with J774A.1 cells and the net accumulation of LDL was monitored by measuring changes in the cellular cholesterol and protein content. Relative to a control sample, cholesterol accumulation was enhanced for aggregate sizes of 75 and 150 nm. The intermediate size aggregates, 100 nm, led to a very striking result demonstrating that cholesterol accumulation was markedly greater than the other samples, and was sufficient to cause cell death. These results underscore an important role of colloidal aggregation, and the influence of LDL aggregate size, in atherosclerosis.     

Effect of captopril on mushroom tyrosinase activity in vitro

The study presented here demonstrates that the antihypertensive drug captopril ([2S]-N-[3-mercapto-2-methylpropionyl]-L-proline) is an irreversible non-competitive inhibitor and an irreversible competitive inhibitor of the monophenolase and diphenolase activities of mushroom tyrosinase when L-tyrosine and L-DOPA were assayed spectrophotometrically in vitro, respectively. Captopril was rendered unstable by tyrosinase catalysis because of the interaction between the enzymatic-generated product (o-quinone) and captopril to give rise to a colourless conjugate. Therefore, captopril was able to prevent melanin formation. The spectrophotometric recordings of the inhibition of tyrosinase by captopril were characterised by the presence of a lag period prior to the attainment of an inhibited steady state rate. The lag period corresponded to the time in which captopril was reacting with the enzymatically generated o-quinone. Increasing captopril concentrations provoked longer lag periods as well as a concomitant decrease in the tyrosinase activity. Both lag period and steady state rate were dependent of captopril, substrate and tyrosinase concentrations. The inhibition of both monophenolase and diphenolase activities of tyrosinase by captopril showed positive kinetic co-operativity which arose from the protection of both substrate and o-quinone against inhibition by captopril. Inhibition experiments carried out using a latent mushroom tyrosinase demonstrated that captopril only bound the enzyme at its active site. The presence of copper ions only partially prevented but not reverted mushroom tyrosinase inhibition. This could be due to the formation of both copper-captopril complex and disulphide interchange reactions between captopril and cysteine rich domains at the active site of the enzyme.     

Adsorption behavior of bovine serum albumin on lowly activated anionic exchangers suggests a new strategy for solid-phase proteomics

Diluted solutions of bovine serum albumin (BSA) (e.g., 0.1 mg /mL) do not form detectable protein large aggregates. Using gel-filtration experiments, we determined that a diluted solution of BSA is 97% monomeric BSA and 3% dimeric. The adsorption of this diluted BSA on highly activated anionic exchangers (e,g., having 40 micromol/wet g) keeps this mainly monomeric form. When supports activated with 2 micromol/wet g are used, only dimers become adsorbed to the support, accounting for 100% of the offered BSA. When the diluted BSA solution is offered to very mildly activated anionic exchangers (even only 0.125 micromol/wet g), an unexpected adsorption of most of the BSA on the support was also observed. These very slightly activated supports are only able to adsorb very large proteins or very large protein-protein complexes, larger than BSA dimers. In fact, a rapid cross-linking of the adsorbed BSA with dextran-aldehyde reveals the formation of very large BSA-BSA complexes with molecular mass higher than 500 000 Da, complexes that may be observed for soluble BSA with very high concentrations but are not detectable at 0.1 mg/mL. Moreover, the size of the aggregates strongly depends on the concentration of the ionized groups on the support: the less activated the supports are, the higher the sizes of the complexes. It seems that the interaction of the BSA molecules on the margins of the BSA aggregate with the groups on the support may stabilize the whole protein aggregate, although some components are not interacting with the support. Aggregates could account for more than 40% of the BSA in the solution after 50 h of incubation. However, only these large BSA aggregates were adsorbed in the support.     

Simulations of reversible protein aggregate and crystal structure.

We simulated the structure of reversible protein aggregates as a function of protein surface characteristics, protein-protein interaction energies, and the entropic penalty accompanying the immobilization of protein in a solid phase. These simulations represent an extension of our previous work on kinetically irreversible protein aggregate structure and are based on an explicit accounting of the specific protein-protein interactions that occur within reversible aggregates and crystals. We considered protein monomers with a mixture of hydrophobic and hydrophilic surface regions suspended in a polar solvent; the energetic driving force for aggregation is provided by the burial of solvent-exposed hydrophobic surface area. We analyzed the physical properties of the generated aggregates, including density, protein-protein contact distributions, solvent accessible surface area, porosity, and order, and compared our results with the protein crystallization literature as well as with the kinetically irreversible case. The physical properties of reversible aggregates were consonant with those observed for the irreversible aggregates, although in general, reversible aggregates were more stable energetically and were more crystal-like in their order content than their irreversible counterparts. The reversible aggregates were less dense than the irreversible aggregates, indicating that the increased energetic stability is derived primarily from the optimality rather than the density of the packing in the solid phase. The extent of hydrophobic protein-protein contacts and solvent-exposed surface area within the aggregate phase depended on the aggregation pathway: reversible aggregates tended to have a greater proportion of hydrophobic-hydrophobic contacts and a smaller fraction of hydrophobic solvent-exposed surface area. Furthermore, the arrangement of hydrophobic patches on the protein surface played a major role in the distribution of protein contacts and solvent content. This was readily reflected in the order of the aggregates: the greater the contiguity of the hydrophobic patches on the monomer surface, the less ordered the aggregates became, despite the opportunities for rearrangement offered by a reversible pathway. These simulations have enhanced our understanding of the impact of protein structural motifs on aggregate properties and on the demarcation between aggregation and crystallization.     

Inhibition of amyloid aggregation of bovine serum albumin by sodium dodecyl sulfate at submicellar concentrations

Sodium dodecyl sulfate (SDS), as an anionic surfactant, can induce protein conformational changes. Recent investigations demonstrated different effects of SDS on protein amyloid aggregation. In the present study, the effect of SDS on amyloid aggregation of bovine serum albumin (BSA) was evaluated. BSA transformed to β-sheet-rich amyloid aggregates upon incubation at pH 7.4 and 65°C, as demonstrated by thioflavin T fluorescence, circular dichroism, and transmission electron microscopy. SDS at submicellar concentrations inhibited BSA amyloid aggregation with IC₅₀ of 47.5 μM. The inhibitory effects of structural analogs of SDS on amyloid aggregation of BSA were determined to explore the structure–activity relationship, with results suggesting that both anionic and alkyl moieties of SDS were critical, and that an alkyl moiety with chain length ≥10 carbon atoms was essential to amyloid inhibition. We attributed the inhibitory effect of SDS on BSA amyloid aggregation to interactions between the detergent molecule and the fatty acid binding sites on BSA. The bound SDS stabilized BSA, thereby inhibiting protein transformation to amyloid aggregates. This study reports for the first time that the inhibitory effect of SDS on albumin fibrillation is closely related to its alkyl structure. Moreover, the specific binding of SDS to albumin is the main driving force in amyloid inhibition. This study not only provides fresh insight into the role of SDS in amyloid aggregation of serum albumin, but also suggests rational design of novel antiamyloidogenic reagents based on specific-binding ligands.     

A rapid and direct method for the determination of active site accessibility in proteins based on ESI-MS and active site titrations

We have developed an electrospray ionisation mass spectrometry (ESI-MS) technique that can be applied to rapidly determine the number of intact active sites in proteins. The methodology relies on inhibiting the protein with an active-site irreversible inhibitor and then using ESI-MS to determine the extent of inhibition. We have applied this methodology to a test system: a serine protease, subtilisin Carlsberg, and monitored the extent of inhibition by phenylmethylsulfonyl fluoride (PMSF), an irreversible serine hydrolase inhibitor as a function of the changes in immobilisation and hydration conditions. Two types of enzyme preparation were investigated, lyophilised enzymes and protein-coated microcrystals (PCMC).     

Zinc metalloenzyme properties of active and latent collagenase from rabbit bone.

1. Inhibition of collagenase from rabbit bone cultures by the chelating agents 1,10-phenanthroline and EDTA is almost completely reversed by Zn2+; other metal cations are less effective in reversing the inhibition. Optimal restoration of activity is achieved at Zn2+ concentrations below that of the chelator, but excess of Zn2+ is inhibitory. 2. Prolonged incubation of collagenase with either chelator causes irreversible inactivation. This inactivation is prevented by Zn2+ at the same concentrations needed to reverse the primary inhibition. 3. Collagenase incorporates 65Zn by exchange when incubated with 1,10-phenanthroline and Zn2+ containing this radioactive isotope. The 65Zn2+ can be removed from its binding site in collagenase by 1,10-phenanthroline or EDTA. Irreversible inactivation of collagenase by chelators destroys its ability to incorporate 65Zn2+. 4. Latent collagenase, the inhibited form in which collagenase first appears in culture, behaves similarly to the active enzyme in 65Zn2+-exchange experiments, but is resistant to irreversible inactivation by chelators. 5. It is concluded that collagenase is a zinc metalloenzyme that forms an inactive and unstable apoenzyme on treatment with chelators. The bound inhibitor component of latent collagenase evidently stabilizes the apoenzyme.     

Induced binding of proteins by ammonium sulfate in affinity and ion-exchange column chromatography

In general, proteins bind to affinity or ion-exchange columns at low salt concentrations, and the bound proteins are eluted by raising the salt concentration, changing the solvent pH, or adding competing ligands. Blue-Sepharose is often used to remove bovine serum albumin (BSA) from samples, but when we applied BSA to Blue-Sepharose in 20 mM phosphate, pH 7.0, 50%-60% of the protein flowed through the column; however, complete binding of BSA was achieved by the addition of 2 M ammonium sulfate (AS) to the column equilibration buffer and the sample. The bound protein was eluted by decreasing the AS concentration or by adding 1 M NaCl or arginine. AS at high concentrations resulted in binding of BSA even to an ion-exchange column, Q-Sepharose, at pH 7.0. Thus, although moderate salt concentrations elute proteins from Blue-Sepharose or ion-exchange columns, proteins can be bound to these columns under extreme salting-out conditions. Similar enhanced binding of proteins by AS was observed with an ATP-affinity column.     

Diisopropyl fluorophosphate labeling of sperm-associated proteinases

Proteinase inhibitors have been shown to be capable of preventing various aspects of fertilization. Diisopropyl fluorophosphate (DFP) is an irreversible inhibitor of trypsin-like enzymes that is commercially available in a radiolabeled form. The experiments described herein were designed to determine if DFP would prevent sperm function in live, motile sperm and to identify the sperm proteins bound with DFP. DFP at 5 mM concentrations had no observable effect on sperm motility, but inhibited the penetration of zona-free hamster ova by human sperm (5.5%) compared to controls (33.5%). Acid extracts of motile sperm that had been incubated with radiolabeled DFP and collected by the swim-up procedure demonstrated the presence of radiolabeled DFP, and the autoradiography of the sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels of these extracts localized the uptake of radiolabeled DFP to proteins in the molecular weight region of the proacrosin-acrosin system. Acid-extracted proteinases from semen samples incubated with DFP demonstrated a concentration-dependent inhibition of both esterolytic hydrolysis of benzoyl-arginine ethyl ester on spectrophotometric analysis and proteolytic activity on gelatin SDS-PAGE zymography. DFP-labeled proteins were precipitated by highly specific antibodies to proacrosin. These results demonstrated that DFP is capable of inhibiting sperm function, and that it associates with the proacrosin-acrosin system in live motile sperm.     

Proteoglycans from bovine nasal and articular cartilages. Fractionation of the link proteins by wheat germ agglutinin affinity chromatography

Two forms of link protein, 46 and 51 kDa, are present in proteoglycan aggregates from both bovine nasal and bovine articular cartilages. Studies reported here show that the link proteins bind to concanavalin A, Lens culinaris agglutinin, Ricinus communis agglutinin, soybean agglutinin, and wheat germ agglutinin lectins. When the link proteins are eluted from these lectins with appropriate competing sugars, the 46- and the 51-kDa link proteins elute together and no separation is achieved. However, when the link proteins bound to wheat germ agglutinin are eluted with a 0 to 4 M guanidine hydrochloride linear gradient, a good separation of the 46- and 51-kDa link proteins is achieved. Wheat germ agglutinin affinity chromatography has been used on a preparative scale to isolate the 51-kDa link protein from mature bovine articular cartilage to homogeneity, in amounts sufficient to examine its effect on proteoglycan aggregate size and stability in sedimentation velocity studies. Proteoglycan aggregates were reassembled from proteoglycan monomers and hyaluronate in the absence of link protein, in the presence of both 46- and 51-kDa link proteins, and in the presence of the individual 51-kDa link protein. The sizes of the aggregates were compared in terms of sedimentation coefficients (s(0)20). The stability of the aggregates was compared in terms of the per cent aggregate present at pH 7 and 5. At pH 7, the sedimentation coefficients (s(0)20) of link-free aggregates, aggregates formed with both link proteins, and aggregates formed with 51-kDa link protein were 72, 93, and 112 S, respectively. Thus, the 51-kDa link protein has a pronounced effect on aggregate size. The link-free aggregate was grossly unstable, and only 36% aggregate was present at pH 5. The aggregate formed with both link proteins was effectively stabilized against dissociation and 79% aggregate was present at pH 5. The aggregate formed with 51-kDa link protein was not effectively stabilized against dissociation, and only 60% aggregate was present at pH 5. Thus, despite its pronounced effect on aggregate size, the 51-kDa link protein does not effectively stabilize the proteoglycan aggregate against dissociation. These results suggest that the 51-kDa link protein may selectively increase aggregate size, while the 46-kDa link protein may be required to effectively stabilize the proteoglycan aggregate against dissociation.     

Crowding Activates ClpB and Enhances Its Association with DnaK for Efficient Protein Aggregate Reactivation

Reactivation of intracellular protein aggregates after a severe stress is mandatory for cell survival. In bacteria, this activity depends on the collaboration between the DnaK system and ClpB, which in vivo occurs in a highly crowded environment. The reactivation reaction includes two steps: extraction of unfolded monomers from the aggregate and their subsequent refolding into the native conformation. Both steps might be compromised by excluded volume conditions that would favor aggregation of unstable protein folding intermediates. Here, we have investigated whether ClpB and the DnaK system are able to compensate this unproductive effect and efficiently reactivate aggregates of three different substrate proteins under crowding conditions. To this aim, we have compared the association equilibrium, biochemical properties, stability, and chaperone activity of the disaggregase ClpB in the absence and presence of an inert macromolecular crowding agent. Our data show that crowding i), increases three to four orders of magnitude the association constant of the functional hexamer; ii), shifts the conformational equilibrium of the protein monomer toward a compact state; iii), stimulates its ATPase activity; and iv), favors association of the chaperone with substrate proteins and with aggregate-bound DnaK. These effects strongly enhance protein aggregate reactivation by the DnaK-ClpB network, highlighting the importance of volume exclusion in complex processes in which several proteins have to work in a sequential manner.     

Detrital aggregates in some Iowa lakes and reservoirs

Detrital aggregates in three eutrophic Iowa lakes and four eutrophic Iowa reservoirs were studied with light and scanning electron microscopy to determine if aggregate morphologies and concentrations were similar. Lake and reservoir aggregates were composed of organic and inorganic particles bound together in an organix matrix. Many of the inorganic particles were calcium carbonate. Obvious bacterial and fungal attachment to the aggregates was rare. Aggregate concentrations ranged from 4 to 274 million aggregates per liter. Aggregates smaller than 18 μm diameter dominated the hyperbolic size-frequency distribution of aggregates in all lakes. Reservoir and lake aggregate concentrations did not differ significantly, but mean aggregate concentrations were directly correlated to the mean chlorophyll a concentration of the lakes. These data strongly suggest that detrital aggregate concentrations are influenced by the trophic status of a lake. Journal Paper No. J-8705 of the Iowa Agriculture and Home Economics Experiment Station, Ames, Iowa. Project 2051. Journal Paper No. J-8705 of the Iowa Agriculture and Home Economics Experiment Station, Ames, Iowa. Project 2051.     

Crowding Activates ClpB and Enhances Its Association with DnaK for Efficient Protein Aggregate Reactivation

Reactivation of intracellular protein aggregates after a severe stress is mandatory for cell survival. In bacteria, this activity depends on the collaboration between the DnaK system and ClpB, which in vivo occurs in a highly crowded environment. The reactivation reaction includes two steps: extraction of unfolded monomers from the aggregate and their subsequent refolding into the native conformation. Both steps might be compromised by excluded volume conditions that would favor aggregation of unstable protein folding intermediates. Here, we have investigated whether ClpB and the DnaK system are able to compensate this unproductive effect and efficiently reactivate aggregates of three different substrate proteins under crowding conditions. To this aim, we have compared the association equilibrium, biochemical properties, stability, and chaperone activity of the disaggregase ClpB in the absence and presence of an inert macromolecular crowding agent. Our data show that crowding i), increases three to four orders of magnitude the association constant of the functional hexamer; ii), shifts the conformational equilibrium of the protein monomer toward a compact state; iii), stimulates its ATPase activity; and iv), favors association of the chaperone with substrate proteins and with aggregate-bound DnaK. These effects strongly enhance protein aggregate reactivation by the DnaK-ClpB network, highlighting the importance of volume exclusion in complex processes in which several proteins have to work in a sequential manner.     

Induced binding of proteins by ammonium sulfate in affinity and ion-exchange column chromatography

In general, proteins bind to affinity or ion-exchange columns at low salt concentrations, and the bound proteins are eluted by raising the salt concentration, changing the solvent pH, or adding competing ligands. Blue-Sepharose is often used to remove bovine serum albumin (BSA) from samples, but when we applied BSA to Blue-Sepharose in 20 mM phosphate, pH 7.0, 50%–60% of the protein flowed through the column; however, complete binding of BSA was achieved by the addition of 2 M ammonium sulfate (AS) to the column equilibration buffer and the sample. The bound protein was eluted by decreasing the AS concentration or by adding 1 M NaCl or arginine. AS at high concentrations resulted in binding of BSA even to an ion-exchange column, Q-Sepharose, at pH 7.0. Thus, although moderate salt concentrations elute proteins from Blue-Sepharose or ion-exchange columns, proteins can be bound to these columns under extreme salting-out conditions. Similar enhanced binding of proteins by AS was observed with an ATP-affinity column.     

Theory and experimental method for determining individual kinetic constants of fast-acting, irreversible proteinase inhibitors

A theory and experimental method are presented to characterize the kinetics of fast-acting, irreversible proteinase inhibitors. The theory is based upon formal analysis of the case of an irreversible inhibitor competing with a substrate for the active-site of a proteinase. From this theory, an experimental method is described by which the individual microscopic kinetic constants for the interaction of the inhibitor with the proteinase can be determined. These are, for a two-step inhibition reaction sequence, the equilibrium dissociation constant and the first-order rate constant for inhibition, and, for a one-step inhibition reaction sequence, the second-order rate constant for inhibition. The theory and experimental method were validated by an analysis of the inhibition of trypsin by the two-step synthetic inhibitor p-nitrophenyl p-guanidinobenzoate and the one-step protein inhibitor bovine pancreatic trypsin inhibitor. The substrate used in these experiments is a new, fluorogenic substrate for trypsin-like serine proteinases (Cbz-Ile-Pro-Arg-NH)2-Rhodamine, the synthesis and properties of which are described.     

Effect of heparin on protein aggregation: inhibition versus promotion

The effect of heparin on both native and denatured protein aggregation was investigated by turbidimetry and dynamic light scattering (DLS). Turbidimetric data show that heparin is capable of inhibiting and reversing the native aggregation of bovine serum albumin (BSA), β-lactoglobulin (BLG), and Zn-insulin at a pH near pI and at low ionic strength I; however, the results vary with regard to the range of pH, I, and protein-heparin stoichiometry required to achieve these effects. The kinetics of this process were studied to determine the mechanism by which interaction with heparin could result in inhibition or reversal of native protein aggregates. For each protein, the binding of heparin to distinctive intermediate aggregates formed at different times in the aggregation process dictates the outcome of complexation. This differential binding was explained by changes in the affinity of a given protein for heparin, partly due to the effects of protein charge anisotropy as visualized by electrostatic modeling. The heparin effect can be further extended to include inhibition of denaturing protein aggregation, as seen from the kinetics of BLG aggregation under conditions of thermally induced unfolding with and without heparin.     

DnaJ recruits DnaK to protein aggregates

Thermal stress might lead to protein aggregation in the cell. Reactivation of protein aggregates depends on Hsp100 and Hsp70 chaperones. We focus in this study on the ability of DnaK, the bacterial representative of the Hsp70 family, to interact with different aggregated model substrates. Our data indicate that DnaK binding to large protein aggregates is mediated by DnaJ, and therefore it depends on its affinity for the cochaperone. Mutations in the structural region of DnaK known as the "latch" decrease the affinity of the chaperone for DnaJ, resulting in a defective activity as protein aggregate-removing agent. As expected, the chaperone activity is recovered when DnaJ concentration is raised to overcome the lower affinity of the mutant for the cochaperone, suggesting that a minimum number of aggregate-bound DnaK molecules is necessary for its efficient reactivation. Our results provide the first experimental evidence of DnaJ-mediated recruiting of ATP-DnaK molecules to the aggregate surface.