Hydrogel polymer appears to mimic the performance of the GroEL/GroES molecular chaperone machine

Controlled protein folding/refolding remains a substantial challenge to the biotechnology industry. Robust and adaptable artificial polymer molecular chaperones could make important contributions towards solving this problem. Taking inspiration from the mechanism of the GroEL/GroES molecular chaperone machine, we report the preparation and testing of a selection of cross-linked thermo-responsive hydrogels, one of which is shown to assist quantitative refolding of a stringent unfolded protein substrate (mitochondrial malate dehydrogenase [mMDH]) during temperature cycling between hydrophobic and hydrophilic states. To our knowledge, this is the first hydrogel-only artificial polymer molecular chaperone to be derived, which is also potentially a generic artificial polymer molecular chaperone for use in a folding bioreactor.     

Temperature‐Responsive Mixed‐Shell Polymeric Micelles for the Refolding of Thermally Denatured Proteins

We have fabricated a mixed‐shell polymeric micelle (MSPM) that closely mimics the natural molecular chaperone GroEL? GroES complex in terms of structure and functionality. This MSPM, which possesses a shared PLA core and a homogeneously mixed PEG and PNIAPM shell, is constructed through the co‐assembly of block copolymers poly(lactide‐b‐poly(ethylene oxide) (PLA‐b‐PEG) and poly(lactide)‐b‐poly(N‐isopropylacryamide) (PLA‐b‐PNIPAM). Above the lower critical solution temperature (LCST) of PNIPAM, the MSPM evolves into a core–shell–corona micelle (CSCM), as a functional state with hydrophobic PNIPAM domains on its surface. Light scattering (LS), TEM, and fluorescence and circular dichroism (CD) spectroscopy were performed to investigate the working mechanism of the chaperone‐like behavior of this system. Unfolded protein intermediates are captured by the hydrophobic PNIPAM domains of the CSCM, which prevent harmful protein aggregation. During cooling, PNIPAM reverts into its hydrophilic state, thereby inducing the release of the bound unfolded proteins. The refolding process of the released proteins is spontaneously accomplished by the presence of PEG in the mixed shell. Carbonic anhydrase B (CAB) was chosen as a model to investigate the refolding efficiency of the released proteins. In the presence of MSPM, almost 93 % CAB activity was recovered during cooling after complete denaturation at 70 °C. Further results reveal that this MSPM also works with a wide spectrum of proteins with more‐complicated structures, including some multimeric proteins. Given the convenience and generality in preventing the thermal aggregation of proteins, this MSPM‐based chaperone might be useful for preventing the toxic aggregation of misfolded proteins in some diseases.     

Tertiary DNA structure in the single-stranded hTERT promoter fragment unfolds and refolds by parallel pathways via cooperative or sequential events

The discovery of G-quadruplexes and other DNA secondary elements has increased the structural diversity of DNA well beyond the ubiquitous double helix. However, it remains to be determined whether tertiary interactions can take place in a DNA complex that contains more than one secondary structure. Using a new data analysis strategy that exploits the hysteresis region between the mechanical unfolding and refolding traces obtained by a laser-tweezers instrument, we now provide the first convincing kinetic and thermodynamic evidence that a higher order interaction takes place between a hairpin and a G-quadruplex in a single-stranded DNA fragment that is found in the promoter region of human telomerase. During the hierarchical unfolding or refolding of the DNA complex, a 15-nucleotide hairpin serves as a common species among three intermediates. Moreover, either a mutant that prevents this hairpin formation or the addition of a DNA fragment complementary to the hairpin destroys the cooperative kinetic events by removing the tertiary interaction mediated by the hairpin. The coexistence of the sequential and the cooperative refolding events provides direct evidence for a unifying kinetic partition mechanism previously observed only in large proteins and complex RNA structures. Not only does this result rationalize the current controversial observations for the long-range interaction in complex single-stranded DNA structures, but also this unexpected complexity in a promoter element provides additional justification for the biological function of these structures in cells.     

Reactivity of aqueous Fe(IV) in hydride and hydrogen atom transfer reactions

Oxidation of cyclobutanol by aqueous Fe(IV) generates cyclobutanone in approximately 70% yield. In addition to this two-electron process, a smaller fraction of the reaction takes place by a one-electron process, believed to yield ring-opened products. A series of aliphatic alcohols, aldehydes, and ethers also react in parallel hydrogen atom and hydride transfer reactions, but acetone and acetonitrile react by hydrogen atom transfer only. Precise rate constants for each pathway for a number of substrates were obtained from a combination of detailed kinetics and product studies and kinetic simulations. Solvent kinetic isotope effect for the self-decay of Fe(IV), kH2O/kD2O = 2.8, is consistent with hydrogen atom abstraction from water.     

Fast folding of a four-helical bundle protein

The FK506-FKBP12 binding-domain of the kinase FRAP (FRB) forms a classic up-down four-helical bundle. The folding pathway of this protein has been investigated using a combination of equilibrium and kinetic studies. The native state of the protein is stable with respect to the unfolded state by some 7 kcal mol(-1) at pH 6.0, 10 degrees C. A kinetic analysis of unfolding and refolding rate constants as a function of chemical denaturant concentration suggests that an intermediate state may be populated during folding at low concentrations of denaturant. The presence of this intermediate state is confirmed by refolding experiments performed in the presence of the hydrophobic dye 8-anilinonaphthalene-1 sulfonate (ANS). ANS binds to the partially folded intermediate state populated during the folding of FRB and undergoes a large change in fluorescence that can be detected using stopped-flow techniques. Analysis of the kinetic data suggests that the intermediate state is compact and it may even be a misfolded species that has to partially unfold before it can reach the transition state. Folding and unfolding rate constants in water are approximately 150-200 s(-1) and 0.005-0.06 s(-1), respectively, at neutral pH and 10 degrees C. The folding of FRB is somewhat slower than for other all-helical proteins, probably as a consequence of the formation of a metastable intermediate state. The folding rate constant in the absence of any populated intermediate can be estimated to be 8800 s(-1). Despite the presence of an intermediate state, which effectively slows folding, the protein still folds rapidly with a half-life of 5 ms at 10 degrees C. The dependence of the rate constants on denaturant concentration indicates that the transition state for folding is compact with some 80% of the surface area exposed in the unfolded state buried in the transition state. Data presented for FRB is compared with kinetic data obtained for other all-helical proteins.     

Chromatographic refolding of recombinant human interferon gamma by an immobilized sht GroEL191-345 column

Minichaperone sht GroEL191-345 was covalently coupled to NHS-activated Sepharose Fast Flow gel. Refolding of recombinant human interferon gamma (rhIFN-gamma) was carried out on a chromatographic column packed with immobilized minichaperone. The effects of salt concentration, urea concentration gradient, elution flow rate and protein loading on the refolding efficiency were investigated. The results indicated that immobilized sht GroEL191-345 chromatography was an effective protocol for the refolding of rhIFN-gamma. When loading 100 microl denatured rhIFN-gamma (17.8 mg/ml), the protein mass recovery and total activity obtained in this optimal process reached 74.25% and 6.74 x 10(6)IU/ml, respectively with the immobilized minichaperone column which was reused for 10 times with 25% decrease of renaturation capacity.     

Lysozyme refolding with immobilized GroEL column chromatography

A refolding chromatography with immobilized molecular chaperonin GroEL was studied for the reactivation of denatured-reduced lysozyme. The effect of denaturant concentration (guanidine hydrochloride, 0.1-1.5 M) in the elution buffer, the elution flow-rate, and the loading concentration and volume of the substrate protein on the reactivation yield was studied. All the operating parameters showed minor effects on the recovery yield of lysozyme mass, which remained at 90-100%, but exhibited relatively notable influences on the specific activity of the recovered lysozyme. For example, there existed an optimum denaturant concentration of about 1 M at which the highest yield of specific activity (up to 97%) was obtained. Using the immobilized GroEL column, 3 ml of the lysozyme (1 mg/ml) per batch could be refolded at an overall yield of 81%, which corresponded to a refolding productivity of 54 mg per 1 gel per h. At comparable reactivation yields (over 80%), this value of productivity was over four-times larger as that of the size-exclusion refolding chromatography reported previously (12 mg per 1 gel per h), indicating the advantage of the present system for producing a high throughput in protein refolding operations.     

Simulation studies of protein folding/unfolding equilibrium under polar and nonpolar confinement

We study the equilibrium folding/unfolding thermodynamics of a small globular miniprotein, the Trp cage, that is confined to the interior of a 2 nm radius fullerene ball. The interactions of the fullerene surface are changed from nonpolar to polar to mimic the interior of the GroEL/ES chaperonin that assists proteins to fold in vivo. We find that nonpolar confinement stabilizes the folded state of the protein due to the effects of volume reduction that destabilize the unfolded state and also due to interactions with the fullerene surface. For the Trp cage, polar confinement has a net destabilizing effect that results from the stabilizing confinement and the competitive exclusion effect that keeps the protein away from the surface hydration shell and stronger interactions between charged side chains in the protein and the polar surface that compete against the formation of an ion pair that stabilizes the protein folded state. We show that confinement effects due to volume reduction can be overcome by sequence-specific interactions of the protein side chains with the encapsulating surface. This study shows that there is a complex balance among many competing effects that determine the mechanism of GroEL chaperonin in enhancing the folding rate of polypeptide inside its cavity.     

Trigger factor slows co-translational folding through kinetic trapping while sterically protecting the nascent chain from aberrant cytosolic interactions

The E. coli chaperone trigger factor (TF) interacts directly with nascent polypeptide chains as they emerge from the ribosome exit tunnel. Small protein domains can fold under the cradle created by TF, but the co-translational folding of larger proteins is slowed down by its presence. Because of the great experimental challenges in achieving high spatial and time resolution, it is not yet known whether or not TF alters the folding properties of small proteins and if the reduced rate of folding of larger proteins is the result of kinetic or thermodynamic effects. We show, by molecular simulations employing a coarse-grained model of a series of ribosome nascent-chain complexes, that TF does not alter significantly the co-translational folding process of a small protein G domain but delays that of a large β-galactosidase domain as a result of kinetic trapping of its unfolded ensemble. We demonstrate that this trapping occurs through a combination of three distinct mechanisms: a decrease in the rate of structural rearrangements within the nascent chain, an increase in the effective exit tunnel length due to folding outside the cradle, and entanglement of the nascent chain with TF. We present evidence that this TF-induced trapping represents a trade-off between promoting co-translational folding and sterically shielding the nascent chain from aberrant cytosolic interactions that could lead to its aggregation or degradation.     

Refolding and purification of histidine-tagged protein by artificial chaperone-assisted metal affinity chromatography

This article has proposed an artificial chaperone-assisted immobilized metal affinity chromatography (AC-IMAC) for on-column refolding and purification of histidine-tagged proteins. Hexahistidine-tagged enhanced green fluorescent protein (EGFP) was overexpressed in Escherichia coli, and refolded and purified from urea-solubilized inclusion bodies by the strategy. The artificial chaperone system was composed of cetyltrimethylammonium bromide (CTAB) and β-cyclodextrin (β-CD). In the refolding process, denatured protein was mixed with CTAB to form a protein–CTAB complex. The mixture was then loaded to IMAC column and the complex was bound via metal chelating to the histidine tag. This was followed by washing with a refolding buffer containing β-CD that removed CTAB from the bound protein and initiated on-column refolding. The effect of the washing time (i.e., on-column refolding time) on mass and fluorescence recoveries was examined. Extensive studies by comparison with other related refolding techniques have proved the advantages of AC-IMAC. In the on-column refolding, the artificial chaperone system suppressed protein interactions and facilitated protein folding to its native structure. So, the on-column refolding by AC-IMAC led to 99% pure EGFP with a fluorescence recovery of 80%. By comparison at a similar final EGFP concentration (0.6–0.8 mg/mL), this fluorescence recovery value was not only much higher than direct dilution (14%) and AC-assisted refolding (26%) in bulk solutions, but also superior to its partner, IMAC (60%). The operating conditions would be further optimized to improve the refolding efficiency.     

In Vitro Renaturation of Alkaline Family G/11 Xylanase via a Folding Intermediate: α-Crystallin Facilitates Refolding in an ATP-Independent Manner

In this study, alkaliphilic family G/11 xylanase from alkali-tolerant filamentous fungi Penicillium citrinum MTCC 6489 was used as a model system to gain insight into the molecular aspects of unfolding/refolding of alkaliphilic glycosyl hydrolase protein family. The intrinsic protein fluorescence suggested a putative intermediate state of protein in presence of 2 M guanidium hydrochloride (GdmCl) with an emission maximum of 353 nm. Here we studied the refolding of GdmCl-denatured alkaline xylanase in the presence and the absence of a multimeric chaperone protein α-crystallin to elucidate the molecular mechanism of intramolecular interactions of the alkaliphilic xylanase protein that dictates its extremophilic character. Our results, based on intrinsic tryptophan fluorescence and hydrophobic fluorophore 8-anilino-1- naphthalene sulfonate-binding studies, suggest that α-crystallin formed a complex with a putative molten globule-like intermediate in the refolding pathway of xylanase in an ATP-independent manner. A 2 M GdmCl is sufficient to denature alkaline xylanase completely. The hydrodynamic radius (R_H) of a native alkaline xylanase is 4.0, which becomes 5.0 in the presence of 2 M GdmCl whereas in presence of the higher concentration of GdmCl R_H value was shifted to 100, indicating the aggregation of denatured xylanase. The α-crystallin·xylanase complex exhibited the recovery of functional activity with the extent of ~43%. Addition of ATP to the complex did not show any significant effect on activity recovery of the denatured protein.     

Protein refolding assisted by periodic mesoporous organosilicas

Herein we report a new strategy for protein refolding by taking advantage of the unique surface and pore characteristics of ethylene-bridged periodic mesoporous organosilica (PMO), which can effectively entrap unfolded proteins and assist refolding by controlled release into the refolding buffer. Hen egg white lysozyme was used as a model protein to demonstrate the new method of protein refolding. Through loading of denatured proteins inside uniform mesoporous channels tailored to accommodate individual protein, protein aggregation was minimized, and the folding rate was increased. Poly(ethyleneglycol) (PEG)-triggered continuous release of entrapped denatured lysozyme allowed high-yield refolding with high cumulative protein concentrations. The new method enhances the oxidative refolding of lysozyme (e.g., over 80% refolding yield at about 0.6 mg/mL).     

Pathway-dependent self-assembly of perylene diimide/peptide conjugates in aqueous medium

Most molecular self-assembly strategies involve equilibrium systems, leading to a single thermodynamic product as a result of weak, reversible non-covalent interactions. Yet, strong non-covalent interactions may result in non-equilibrium self-assembly, in which structural diversity is achieved by forming several kinetic products based on a single covalent building block. We demonstrate that well-defined amphiphilic molecular systems based on perylene diimide/peptide conjugates exhibit kinetically controlled self-assembly in aqueous medium, enabling pathway-dependent assembly sequences, in which different organic nanostructures are evolved in a stepwise manner. The self-assembly process was characterized using UV/Vis circular dichroism (CD) spectroscopy, and cryogenic transmission electron microscopy (cryo-TEM). Our findings show that pathway-controlled self-assembly may significantly broaden the methodology of non-covalent synthesis.     

Mechanistic insight into the activity of tyrosinase from variable-temperature studies in an aqueous/organic solvent

The activity of mushroom tyrosinase towards a representative series of phenolic and diphenolic substrates structurally related to tyrosine has been investigated in a mixed solvent of 34.4% methanol-glycerol (7:1, v/v) and 65.6% (v/v) aqueous 50 mM Hepes buffer at pH 6.8 at various temperatures. The kinetic activation parameters controlling the enzymatic reactions and the thermodynamic parameters associated with the process of substrate binding to the enzyme active species have been deduced from the temperature variation of the kcat and KM parameters. The activation free energy is dominated by the enthalpic term, the value of which lies in the relatively narrow range of 61+/-9 kJ mol(-1) irrespective of substrate or reaction type (monophenolase or diphenolase). The activation entropies are small and generally negative and contribute no more than 10% to the activation free energy. The substrate binding parameters are characterized by large and negative enthalpy and entropy contributions, which are typically dictated by polar protein-substrate interactions. The substrate 4-hydroxyphenylpropionic acid exhibits a strikingly anomalous temperature dependence of the enzymatic oxidation rate, with deltaH(double dagger) approximately = 150 kJ mol(-1) and deltaS(double dagger) approximately = 280 J K(-1) mol(-1), due to the fact that it can competitively bind to the enzyme through the phenol group, like the other substrates, or the carboxylate group, like carboxylic acid inhibitors. A kinetic model that takes into account the dual substrate/inhibitor nature of this compound enables rationalization of this anomalous behavior.     

Determinations cinetiques par analyse microcalorimetrique differentielle. XVI. Decomposition du percarbonate de O,O-t-butyle et O-isopropenyle en solution

Kinetic studies by differential scanning microcalorimetry have shown that the free radical decomposition of O,O-t-butyl and O-isopropenylperoxycarbonate is induced, to a great extent, by the addition of free radicals to the vinylic double bond. If the solvent can give stabilized free radicals, then acetonyl radicals add themselves to the double bond leading to acetonylacetone; in this case, the kinetic study allows the characteristic parameters of the thermal stability of the peroxycarbonate to be determined. When the radicals issued from the solvent can add themselves to the double bond, the induced decomposition finds expression in the acetonylation of the solvent; as for kinetics, such an enhancement of the reaction rate takes place that the “spontaneous” homolytic decomposition takes only a minor part even for very low concentrations of the peroxycarbonate solutions.     

Toward an understanding of the mechanisms of the intramolecular

The molecular mechanism for the intramolecular [5 + 2] cycloaddition reaction of beta-silyloxy-gamma-pyrones bearing tethered alkenes has been characterized using ab initio methods. A comparative study for this sort of cycloaddition carried out at different computational levels points out that the B3LYP/6-31G calculations give similar barriers to those obtained with the MP3/6-31G level. Analysis of the energetic results shows that the reaction takes place along a stepwise process: first, the migration of the neighboring silyl group to the carbonyl group of the gamma-pyrone takes place to give a weak oxidopyrylium ylide intermediate, which by a subsequent concerted intramolecular [5 + 2] cycloaddition affords the final cycloadduct. The cycloaddition process is very stereoselective due to the constraints imposed by the tether. The [5 + 2] cycloaddition reaction has a large barrier, and the presence of the silyloxy group and the intramolecular character of the process are necessary to ensure the thermodynamic and kinetic feasibility of these cycloadditions.     

Thermodynamic and kinetic factors in the hydrothermal synthesis of hybrid frameworks: zinc 4-cyclohexene-1,2-dicarboxylates

Experimental and computational studies indicate that the formation of a series of zinc 4-cyclohexene-1,2-dicarboxylates takes place under thermodynamic rather than kinetic control.     

Solvolysis of benzoyl halides in water/NH4DEHP/isooctane microemulsions

A study was carried out on the solvolysis reactions of different benzoyl halides in microemulsions of water/NH4DEHP/isooctane, where NH4DEHP is ammonium bis(2-ethylhexyl) phosphate. Because of the low solubility of benzoyl halides in water, they are distributed between the continuous medium and the interface of the microemulsion, where the reaction takes place. The application of the pseudophase model has allowed us to obtain the distribution constants and the rate constants at the interface for the benzoyl halides. Reaction mechanisms and the changes in these mechanisms in terms of the water content of the microemulsion have been determined on the basis of kinetic data. The influence of the substituent and the leaving group on the reaction rate has been investigated. A comparison of kinetic results with those previously obtained in water/AOT/isooctane microemulsions allows a kinetic evaluation of the change in the microemulsion properties with the surfactant.