Molecular simulation of surfactant-assisted protein refolding

Protein refolding to its native state in vitro is a challenging problem in biotechnology, i.e., in the biomedical, pharmaceutical, and food industry. Protein aggregation and misfolding usually inhibit the recovery of proteins with their native states. These problems can be partially solved by adding a surfactant into a suitable solution environment. However, the process of this surfactant-assisted protein refolding is not well understood. In this paper, we wish to report on the first-ever simulations of surfactant-assisted protein refolding. For these studies, we defined a simple model for the protein and the surfactant and investigated how a surfactant affected the folding behavior of a two-dimensional lattice protein molecule. The model protein and model surfactant were chosen such that we could capture the important features of the folding process and the interaction between the protein and the surfactant, namely, the hydrophobic interaction. It was shown that, in the absence of surfactants, a protein in an "energy trap" conformation, i.e., a local energy minima, could not fold into the native form, which was characterized by a global energy minimum. The addition of surfactants created folding pathways via the formation of protein-surfactant complexes and thus enabled the conformations that fell into energy trap states to escape from these traps and to form the native proteins. The simulation results also showed that it was necessary to match the hydrophobicity of surfactant to the concentration of denaturant, which was added to control the folding or unfolding of a protein. The surfactants with different hydrophobicity had their own concentration range on assisting protein refolding. All of these simulations agreed well with experimental results reported elsewhere, indicating both the validity of the simulations presented here and the potential application of the simulations for the design of a surfactant on assisting protein refolding.     

Dynamic redox environment-intensified disulfide bond shuffling for protein refolding in vitro: molecular simulation and experimental validation

One challenge in protein refolding is to dissociate the non-native disulfide bonds and promote the formation of native ones. In this study, we present a coarse-grained off-lattice model protein containing disulfide bonds and simulate disulfide bond shuffling during the folding of this model protein. Introduction of disulfide bonds in the model protein led to enhanced conformational stability but reduced foldability in comparison to counterpart protein without disulfide bonds. The folding trajectory suggested that the model protein retained the two-step folding mechanism in terms of hydrophobic collapse and structural rearrangement. The disulfide bonds located in the hydrophobic core were formed before the collapsing step, while the bonds located on the protein surface were formed during the rearrangement step. While a reductive environment at the initial stage of folding favored the formation of native disulfide bonds in the hydrophobic core, an oxidative environment at a later stage of folding was required for the formation of disulfide bonds at protein surface. Appling a dynamic redox environment, that is, one that changes from reductive to oxidative, intensified disulfide bond shuffling and thus resulted in improved recovery of the native conformation. The above-mentioned simulation was experimentally validated by refolding hen-egg lysozyme at different urea concentrations and oxidized glutathione/reduced glutathione (GSSG/GSH) ratios, and an optimal redox environment, in terms of the GSSG to GSH ratio, was identified. The implementation of a dynamic redox environment by tuning the GSSG/GSH ratio further improved the refolding yield of lysozyme, as predicted by molecular simulation.     

De novo refolding and aggregation of insulin in a nonaqueous environment: an inside out protein remake

While thermodynamic penalties associated with protein-water interactions are the key driving force of folding, perturbed hydration of destabilized protein molecules may trigger aggregation, which in vivo often causes cellular and histological damage. Here we show, that the denatured state of an alpha-helical protein, insulin, converts to a non-native beta-sheet-rich structure upon de novo "refolding" in an anhydrous environment. The beta-pleated conformer precipitates from solutions of DMSO-denatured insulin upon dilution with chloroform. DMSO destroys hydrogen bond network of the native protein acting as a strong acceptor of main chain hydrogen bonds. Upon the addition of chloroform, which is a weak hydrogen bond donor per se, competitive hydrogen bonds between DMSO and chloroform are formed. This leads to the release of unfolded insulin molecules. In the absence of water, the imminent saturation of polypeptide's dandling hydrogen bonds does not produce the native and predominantly alpha-helical state but a beta-sheet-rich structure, which is morphologically and spectrally distinct from insulin amyloid fibrils. Unlike insulin fibrils, the beta-sheet conformer is metastable and refolds spontaneously to the native form in an aqueous environment. This implies that "folding" in the absence of water results in inefficient burial of hydrophobic side-chains, and thermodynamic frustration at the water-protein interface.     

Mechanism of simultaneously refolding and purification of proteins by hydrophobic interaction chromatographic unit and applications

The hydrophobic amino acid residues of a denatured protein molecule tend to react with the particles of the stationary phase of hydrophobic interaction chromatography (STHIC). These hydrophobic interactions prevent the denatured protein molecules from aggregating with each other. The STHIC can provide high enough energy to a denatured protein molecule to make it dehydration and to refold it into its native or various intermediate states. The outcome not only depends on the specific interactions between amino acids, the structure of STHIC, but also depends on the association between the STHIC and mobile phase. The mechanism of protein refolding and the principle of its quality control by HPHIC were also presented. By appropriate selection of the chromatographic condition, several denatured proteins can be refolded and separated simultaneously in a single chromatographic run. A specially designed unit, with diameter much larger than its length, was designed and employed for both laboratory and preparative     

Interplay of secondary structures and side-chain contacts in the denatured state of BBA1

The denatured state of a miniprotein BBA1 is studied under the native condition with the AMBER/Poisson-Boltzmann energy model and with the self-guided enhanced sampling technique. Forty independent trajectories are collected to sample the highly diversified denatured structures. Our simulation data show that the denatured BBA1 contains high percentage of native helix and native turn, but low percentage of native hairpin. Conditional population analysis indicates that the native helix formation and the native hairpin formation are not cooperative in the denatured state. Side-chain analysis shows that the native hydrophobic contacts are more preferred than the non-native hydrophobic contacts in the denatured BBA1. In contrast, the salt-bridge contacts are more or less nonspecific even if their populations are higher than those of hydrophobic contacts. Analysis of the trajectories shows that the native helix mostly initiates near the N terminus and propagates to the C terminus, and mostly forms from 3(10)-helix/turn to alpha helix. The same analysis shows that the native turn is important but not necessary in its formation in the denatured BBA1. In addition, the formations of the two strands in the native hairpin are rather asymmetric, demonstrating the likely influence of the protein environment. Energetic analysis shows that the native helix formation is largely driven by electrostatic interactions in denatured BBA1. Further, the native helix formation is associated with the breakup of non-native salt-bridge contacts and the accumulation of native salt-bridge contacts. However, the native hydrophobic contacts only show a small increase upon the native helix formation while the non-native hydrophobic contacts stay essentially the same, different from the evolution of hydrophobic contacts observed in an isolated helix folding.     

KOH catalysed preparation of activated carbon aerogels for dye adsorption

Surfactants prevent the irreversible aggregation of partially refolded proteins, and they are also known to assist in protein refolding. A novel approach to protein refolding that utilizes a pair of low molecular weight folding assistants, a detergent and cyclodextrin, was proposed by Rozema and Gellman (D. Rozema, S.H. Gellman, J. Am. Chem. Soc. 117 (1995) 2373). We report the refolding of bovine serum albumin (BSA) assisted by these artificial chaperones, utilizing gemini surfactants for the first time. A combination of cationic gemini surfactants, bis(cetyldimethylammonium)pentane dibromide (C(16)H(33)(CH(3))(2)N(+)-(CH(2))(5)-N(+)(CH(3))(2)C(16)H(33)·2Br(-) designated as G5 and bis(cetyldimethylammonium)hexane dibromide (C(16)H(33)(CH(3))(2)N(+)-(CH(2))(6)-N(+)(CH(3))(2)C(16)H(33)·2Br(-) designated as G6 and cyclodextrins, was used to refold guanidinium chloride (GdCl) denatured BSA in the artificial chaperone assisted two step method. The single chain cationic surfactant cetyltrimethylammonium bromide (CTAB) was used for comparative studies. The studies were carried out in an aqueous medium at pH 7.0 using circular dichroism, dynamic light scattering and ANS binding studies. The denatured BSA was found to get refolded by very small concentrations of gemini surfactant at which the single chain counterpart was found to be ineffective. Different from the single chain surfactant, the gemini surfactants exhibit much stronger electrostatic and hydrophobic interactions with the protein and are thus effective at much lower concentrations. Based on the present study it is expected that gemini surfactants may prove useful in the protein refolding operations and may thus be effectively employed to circumvent the problem of misfolding and aggregation.     

Continuous matrix-assisted refolding of proteins

A refolding reactor was developed for continuous matrix-assisted refolding of proteins. The reactor was composed of an annular chromatography system and an ultrafiltration system to recycle aggregated proteins produced during the refolding reaction. The feed solution containing the denatured protein was continuously fed to the rotating bed perfused with buffer promoting folding of the protein. As the protein passed through the column, it was separated from chaotropic and reducing agents and the refolding process took place. Native proteins and aggregates could be continuously separated due to different molecular size. The exit stream containing aggregates was collected, concentrated by ultrafiltration and recycled to the feed solution. The high concentrations of chaotropic and reducing agents in the feed solution enabled dissociation of the recycled aggregates and consequently were fed again to the refolding reactor. When the initial feed mixture of denatured protein is used up, only buffer-containing chaotropic agents and recycled aggregates are fully converted to native protein. This process resulted in a stoichiometric conversion from the denatured protein to its correctly folded native state. The system was tested with bovine alpha-lactalbumin as model protein. Superdex 75 PrepGrade was used as size-exclusion medium. The yield of 30% active monomer in the batch process was improved to 41% at a recycling rate of 65%. Assuming that the aggregates can be redissolved and recycled into the feed stream in a quantitative manner, a refolding yield close to 100% is possible. The method can be also applied to other chromatographic principles suited for the separation of aggregates.     

Understanding the folding rates and folding nuclei of globular proteins

The first part of this paper contains an overview of protein structures, their spontaneous formation ("folding"), and the thermodynamic and kinetic aspects of this phenomenon, as revealed by in vitro experiments. It is stressed that universal features of folding are observed near the point of thermodynamic equilibrium between the native and denatured states of the protein. Here the "two-state" ("denatured state" <--> "native state") transition proceeds without accumulation of metastable intermediates, but includes only the unstable "transition state". This state, which is the most unstable in the folding pathway, and its structured core (a "nucleus") are distinguished by their essential influence on the folding/unfolding kinetics. In the second part of the paper, a theory of protein folding rates and related phenomena is presented. First, it is shown that the protein size determines the range of a protein's folding rates in the vicinity of the point of thermodynamic equilibrium between the native and denatured states of the protein. Then, we present methods for calculating folding and unfolding rates of globular proteins from their sizes, stabilities and either 3D structures or amino acid sequences. Finally, we show that the same theory outlines the location of the protein folding nucleus (i.e., the structured part of the transition state) in reasonable agreement with experimental data.     

Folding Dynamics of 10‐Residue β‐Hairpin Peptide Chignolin

Short peptides that fold into β‐hairpins are ideal model systems for investigating the mechanism of protein folding because their folding process shows dynamics typical of proteins. We performed folding, unfolding, and refolding molecular dynamics simulations (total of 2.7 μs) of the 10‐residue β‐hairpin peptide chignolin, which is the smallest β‐hairpin structure known to be stable in solution. Our results revealed the folding mechanism of chignolin, which comprises three steps. First, the folding begins with hydrophobic assembly. It brings the main chain together; subsequently, a nascent turn structure is formed. The second step is the conversion of the nascent turn into a tight turn structure along with interconversion of the hydrophobic packing and interstrand hydrogen bonds. Finally, the formation of the hydrogen‐bond network and the complete hydrophobic core as well as the arrangement of side‐chain–side‐chain interactions occur at approximately the same time. This three‐step mechanism appropriately interprets the folding process as involving a combination of previous inconsistent explanations of the folding mechanism of the β‐hairpin, that the first event of the folding is formation of hydrogen bonds and the second is that of the hydrophobic core, or vice versa.     

The interplay of turn formation and hydrophobic interactions on the early kinetic events in protein folding

While both turn formation and hydrophobic interactions play dominant roles in the initiation of protein folding, their individual contributions to the folding kinetics and to the structural stability of the protein still remain poorly understood. Here, we applied a photolabile linker to "cage" some important structural motifs, including both α-helices and β-sheets, into their non-native states. These "caged" structural motifs are then relaxed by laser-flash photolysis and their refolding events followed by photoacoustic calorimetry (PAC) and photothermal beam deflection (PBD). These experiments, combined with our previous results, revealed that spontaneous α-helix formation can occur extremely rapidly (10(8)-10(9) s(-1)) if the process is driven solely by turn formation followed by helix propagation. However, if sequestering of the side chains of hydrophobic amino acid residues participates in the refolding process, which may provide additional driving force beyond that afforded by turn formation alone, the refolding rate will be retarded, often by many orders of magnitude. This is usually the case in the formation of three-stranded β-sheets (10(7)-10(8) s(-1)) and β-hairpins (10(5)-10(6) s(-1)). Thus, we propose that proteins take advantage of the hierarchy of timescales associated with either turn formation, hydrophobic interactions, or global collapse of tertiary structure to accomplish the folding process in an orderly fashion, as these events are sufficiently separated in time and do not interfere with one another.     

EFFECT OF SURFACTANTS ON THE PREVENTION OF PROTEIN AGGREGATION DURING UNFOLDING AND REFOLDING PROCESSES-COMPARISON WITH MOLECULAR CHAPERONE α -CRYSTALLIN

Molecular chaperones prevent protein aggregation. Understanding of their mechanism of actions remains obscured by the complexity of their three dimensional structure. Surfactants are much simpler molecules and can also inhibit protein aggregation. In the present work, prevention of aggregation by ionic and non-ionic surfactants during thermal and non-thermal unfolding and refolding of a number of proteins are studied. Attempts have been made to compare the dose dependence of surfactants with that of molecular chaperone a-crystallin in preventing various protein aggregations. Conformational features of proteins whose aggregation have been prevented by surfactants and a-crystallin are compared with each other. It has been found that in terms of conformation of the substrate protein, chaperones restrict the bound protein in a conformation not far from that of the native protein, whereas ionic surfactants may lead to considerably different substrate protein conformation. Non-ionic surfactants, though slow in solubilising protein aggregates, do not cause great changes in protein conformation.     

A temperature-jump stopped-flow system for monitoring chemical kinetics triggered by rapid cooling

This work reports the implementation of a stopped-flow system for studying the kinetics of protein folding triggered by rapid cooling. The transition from denaturing temperatures to ambient conditions is achieved by rapid mixing of pre-heated protein solution with cold refolding buffer. The estimated dead-time of the apparatus is 8 ms. Folding kinetics are monitored by fluorescence spectroscopy. Careful tuning of the solution mixing ratio is required for the elimination of baseline artifacts that could mask fluorescence signals originating from protein conformational changes. The viability of the rapid cooling method is demonstrated by applying it to monitor the refolding of thermally denatured cytochrome c. Following a heat exposure time of 3 min, the protein shows multi-exponential folding kinetics, ultimately resulting in a fluorescence level that is virtually indistinguishable from that of the native state. In contrast, incomplete refolding is observed when the heat exposure time is extended to 30 min. This effect may be due to aggregation phenomena affecting the thermally denatured protein. It appears that the rapid cooling approach reported in this work may become a useful tool for temperature-jump studies in various areas of chemistry and biochemistry.     

Molecular simulation of polymer assisted protein refolding

Protein refolding in vitro, the formation of the tertiary structure that enables the protein to display its biological function, can be significantly enhanced by adding a polymer of an appropriate hydrophobicity and concentration into the refolding buffer. A molecular simulation of the refolding of a two-dimensional simple lattice protein was presented. A protein folding map recording the occurrence frequency of specified conformations was derived, from which the refolding thermodynamics and kinetics were interpreted. It is shown that, in the absence of polymer, the protein falls into the "energy trapped" conformations characterized by a high intramolecular hydrophobic interaction, denoted as HH contact, and a high magnitude of the structure overlap function, chi. This makes it difficult for the protein to fold to the native state. The polymer with a suitable chain length, concentration, and hydrophobicity has formed complex with partially folded protein and created diversified intermediates with low chi. This gives more pathways for the protein to fold to the native state. At a given hydrophobicity, the short chain polymer has a broader concentration range where it assists protein folding than those of long chains. The above simulation agrees well with the experimental results reported elsewhere [Cleland et al., J. Biol. Chem. 267, 13327 (1992); ibid., Bio/Technology 10, 1013 (1992); Chen et al., Enzyme Microb. Technol. 32, 120 (2003); Lu et al., Biochem. Eng. J. 24, 55 (2005); ibid., J. Chem. Phys. 122, 134902 (2005); ibid., Biochem. Eng. J. (to be published)] and is of fundamental importance for the design and application of polymers for protein refolding.     

Folding transition-state and denatured-state ensembles of FSD-1 from folding and unfolding simulations

Characterization of the folding transition-state ensemble and the denatured-state ensemble is an important step toward a full elucidation of protein folding mechanisms. We report herein an investigation of the free-energy landscape of FSD-1 protein by a total of four sets of folding and unfolding molecular dynamics simulations with explicit solvent. The transition-state ensemble was initially identified from unfolding simulations at 500 K and was verified by simulations at 300 K starting from the ensemble structures. The denatured-state ensemble and the early-stage folding were studied by a combination of unfolding simulations at 500 K and folding simulations at 300 K starting from the extended conformation. A common feature of the transition-state ensemble was the substantial formation of the native secondary structures, including both the alpha-helix and beta-sheet, with partial exposure of the hydrophobic core in the solvent. Both the native and non-native secondary structures were observed in the denatured-state ensemble and early-stage folding, consistent with the smooth experimental melting curve. Interestingly, the contact orders of the transition-state ensemble structures were similar to that of the native structure and were notably lower than those of the compact structures found in early-stage folding, implying that chain and topological entropy might play significant roles in protein folding. Implications for FSD-1 folding mechanisms and the rate-limiting step are discussed. Analyses further revealed interesting non-native interactions in the denatured-state ensemble and early-stage folding and the possibility that destabilization of these interactions could help to enhance the stability and folding rate of the protein.     

Thermodynamic and functional properties of legumin (11S globulin from Vicia faba) in the presence of small-molecule surfactants: effect of temperature and pH

We report on the effect of a set of water-dispersible small-molecule surfactants (the main and the longest-hydrocarbon components of which are a citric acid ester of monostearate, a sodium salt of stearol-lactoyl lactic acid, and a polyglycerol ester of stearic acid) on molecular, thermodynamic, and functional properties of the major storage protein of broad beans (Vicia faba) legumin in different molecular states (native, heated, and acid-denatured). The interaction between legumin and the surfactants has been characterized by a combination of thermodynamic methods, namely, mixing calorimetry and multiangle laser static and dynamic light scattering. It was found that hydrogen bonds, electrostatic interactions, and hydrophobic contacts provided a basis for the interactions between the surfactants and both the native and the denatured protein in aqueous medium. Intensive association of the protein molecules in a bulk aqueous medium in the presence of the surfactants was revealed by static and dynamic laser light scattering. In consequence of this, both the surface activity and the gel-forming ability of legumin increased markedly, which has been shown by tensiometry, estimation of protein foaming capacity, and steady-state viscometry. A likely molecular mechanism underlying the effects of small-molecule surfactants on legumin structure-forming properties at the interface and in a bulk aqueous medium is discussed.     

Folding and assembly of hemoglobin monitored by electrospray mass spectrometry using an on-line dialysis system

The native structure of hemoglobin (Hb) comprises two alpha- and two beta-subunits, each of which carries a heme group. There appear to be no previous studies that report the in vitro folding and assembly of Hb from highly unfolded alpha- and beta-globin in a "one-pot" reaction. One difficulty that has to be overcome for studies of this kind is the tendency of Hb to aggregate during refolding. This work demonstrates that denaturation of Hb in 40% acetonitrile at pH 10.0 is reversible. A dialysis-mediated solvent change to a purely aqueous environment of pH 8.0 results in Hb refolding without any apparent aggregation. Fluorescence, Soret absorption, circular dichroism, and ESI mass spectra of the protein recorded before unfolding and after refolding are almost identical. By employing an externally pressurized dialysis cell that is coupled on-line to an ESI mass spectrometer, changes in heme binding behavior, protein conformation, and quaternary structure can be monitored as a function of time. The process occurs in a stepwise sequential manner, leading from monomeric alpha- and beta-globin to heterodimeric species, which then assemble into tetramers. Overall, this mechanism is consistent with previous studies employing the mixing of folded alpha- and beta-globin. However, some unexpected features are observed, e.g., a heme-deficient beta-globin dimer that represents an off-pathway intermediate. Monomeric beta-globin is capable of binding heme before forming a complex with an alpha-subunit. This observation suggests that holo-alpha-apo-beta globin does not represent an obligatory intermediate during Hb assembly, as had been proposed previously. The on-line dialysis/ESI-MS approach developed for this work represents a widely applicable tool for studying the folding and self-assembly of noncovalent biological complexes.     

Effects of turn stability on the kinetics of refolding of a hairpin in a beta-sheet

As part of our continuing study of the effects of the turn sequence on the conformational stability as well as the mechanism of folding of a beta-sheet structure, we have undertaken a parallel investigation of the solution structure, conformational stability, and kinetics of refolding of the beta-sheet VFIVDGOTYTEV(D)PGOKILQ. The latter peptide is an analogue of the original Gellman beta-sheet VFITS(D)PGKTYTEV(D)PGOKILQ, wherein the TS(D)PGK turn sequence in the first hairpin has been replaced by VDGO. Thermodynamics studies revealed comparable conformational stability of the two peptides. However, unlike the Gellman peptide, which showed extremely rapid refolding of the first hairpin, early kinetic events associated with the refolding of the corresponding hairpin in the VDGO mutant were found to be significantly slower. A detailed study of the conformation of the modified peptide suggested that hydrophobic interactions might be contributing to its stability. Accordingly, we surmise that the early kinetic events are sensitive to whether the formation of the hairpin is nucleated at the turn or by sequestering of the hydrophobic residues across the strand, before structural rearrangements to produce the nativelike topology. Nucleation of the hairpin at the turn is expected to be intrinsically rapid for a strong turn. However, if the process must involve collapse of hydrophobic side chains, the nucleation should be slower as solvent molecules must be displaced to sequester the hydrophobic residues. These findings reflect the contribution of different forces toward nucleation of hairpins in the mechanism of folding of beta-sheets.     

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).     

A minimal proteinlike lattice model: an alpha-helix motif

A simple protein model of a four-helix bundle motif on a face-centered cubic lattice has been studied. Total energy of a conformation includes attractive interactions between hydrophobic residues, repulsive interactions between hydrophobic and polar residues, and a potential that favors helical turns. Using replica exchange Monte Carlo simulations we have estimated a set of parameters for which the native structure is a global minimum of conformational energy. Then we have shown that all the above types of interactions are necessary to guarantee the cooperativity of folding transition and to satisfy the thermodynamic hypothesis.