Refolding Intermediates Research Articles

Intermediates in SARS-CoV-2 spike-mediated cell entry.

SARS-CoV-2 cell entry is completed after viral spike (S) protein–mediated membrane fusion between viral and host cell membranes. Stable prefusion and postfusion S structures have been resolved by cryo–electron microscopy and cryo–electron tomography, but the refolding intermediates on the fusion pathway are transient and have not been examined. We used an antiviral lipopeptide entry inhibitor to arrest S protein refolding and thereby capture intermediates as S proteins interact with hACE2 and fusion-activating proteases on cell-derived target membranes. Cryo–electron tomography imaged both extended and partially folded intermediate states of S2, as well as a novel late-stage conformation on the pathway to membrane fusion. The intermediates now identified in this dynamic S protein–directed fusion provide mechanistic insights that may guide the design of CoV entry inhibitors.

Grp94 Works Upstream of BiP in Protein Remodeling Under Heat Stress

Hsp90 and Hsp70 are highly conserved molecular chaperones that promote the proper folding and activation of substrate proteins that are often referred to as clients. The two chaperones functionally collaborate to fold specific clients in an ATP-dependent manner. In eukaryotic cytosol, initial client folding is done by Hsp70 and its co-chaperones, followed by a direct transfer of client refolding intermediates to Hsp90 for final client processing. However, the mechanistic details of collaboration of organelle specific Hsp70 and Hsp90 are lacking. This work investigates the collaboration of the endoplasmic reticulum (ER) Hsp70 and Hsp90, BiP and Grp94 respectively, in protein remodeling using in vitro refolding assays. We show that under milder denaturation conditions, BiP collaborates with its co-chaperones to refold misfolded proteins in an ATP-dependent manner. Grp94 does not play a major role in this refolding reaction. However, under stronger denaturation conditions that favor aggregation, Grp94 works in an ATP-independent manner to bind and hold misfolded clients in a folding competent state for subsequent remodeling by the BiP system. We also show that the collaboration of Grp94 and BiP is not simply a reversal of the eukaryotic refolding mechanism since a direct interaction of Grp94 and BiP is not required for client transfer. Instead, ATP binding but not hydrolysis by Grp94 facilitates the release of the bound client, which is then picked up by the BiP system for subsequent refolding in a Grp94-independent manner.

Disulfide bond analysis of toxin refolding intermediates by mass spectrometry

Disulfide bond analysis of toxin refolding intermediates by mass spectrometry

Immobilization of inulinase from Aspergillus niger on octadecyl substituted nanoporous silica: Inulin hydrolysis in a continuous mode operation

Abstract Inulinase hydrolyzes inulin and produce high fructose syrup in a single-step reaction. However, finding the best working conditions and the highest inulinase stability, especially in continuous mode operations are of critical value. Native (endo-inulinase (EC 3.2.1.7)) and chemically modified inulinases (endo-inulinase stabilized with pyridoxal 5′-phosphate/ascorbic acid in a semi-rational chemical modification approach) were immobilized on octadecyl substituted nanoporous silica (with the average pore size of 66 nm) by using dioxane to extend the hydrophobic interactions between inulinase refolding intermediates and the hydrophobic alkyl moieties of the support. Native and modified inulinases revealed their maximum surface hydrophobicity at 40% and 50% dioxane in water, respectively. Immobilized inulinases (native and modified) showed 7.89 and 3.73 fold increase in their half lives compared to the free native sample., higher reusability compared to free inulinases (native and modified) and also could retain their activities after 10 hydrolysis cycles in a continuous mode of experimental conditions.

Reversible Interactions of Proteins with Mixed Shell Polymeric Micelles: Tuning the Surface Hydrophobic/Hydrophilic Balance toward Efficient Artificial Chaperones.

Molecular chaperones can elegantly fine-tune its hydrophobic/hydrophilic balance to assist a broad spectrum of nascent polypeptide chains to fold properly. Such precious property is difficult to be achieved by chaperone mimicking materials due to limited control of their surface characteristics that dictate interactions with unfolded protein intermediates. Mixed shell polymeric micelles (MSPMs), which consist of two kinds of dissimilar polymeric chains in the micellar shell, offer a convenient way to fine-tune surface properties of polymeric nanoparticles. In the current work, we have fabricated ca. 30 kinds of MSPMs with finely tunable hydrophilic/hydrophobic surface properties. We investigated the respective roles of thermosensitive and hydrophilic polymeric chains in the thermodenaturation protection of proteins down to the molecular structure. Although the three kinds of thermosensitive polymers investigated herein can form collapsed hydrophobic domains on the micellar surface, we found distinct capability to capture and release unfolded protein intermediates, due to their respective affinity for proteins. Meanwhile, in terms of the hydrophilic polymeric chains in the micellar shell, poly(ethylene glycol) (PEG) excels in assisting unfolded protein intermediates to refold properly via interacting with the refolding intermediates, resulting in enhanced chaperone efficiency. However, another hydrophilic polymer-poly(2-methacryloyloxyethyl phosphorylcholine) (PMPC) severely deteriorates the chaperone efficiency of MSPMs, due to its protein-resistant properties. Judicious combination of thermosensitive and hydrophilic chains in the micellar shell lead to MSPM-based artificial chaperones with optimal efficacy.

Classification of chemical chaperones based on their effect on protein folding landscapes.

Various small molecules present in biological systems can assist protein folding in vitro and are known as chemical chaperones. De novo design of chemical chaperones with higher activity than currently known examples is desirable to ameliorate protein misfolding and aggregation in multiple contexts. However, this development has been hindered by limited knowledge of their activities. It is thought that chemical chaperones are typically poor solvents for a protein backbone and hence facilitate native structure formation. However, it is unknown if different chemical chaperones can act differently to modulate folding energy landscapes. Using a model slow folding protein, double-mutant Maltose-binding protein (DM-MBP), we show that a canonical chemical chaperone, trimethylamine-N-oxide (TMAO), accelerates refolding by decreasing the flexibility of the refolding intermediate (RI). Among a number of small molecules that chaperone DM-MBP folding, proline and serine stabilize the transition state (TS) enthalpically, while trehalose behaves like TMAO and increases the rate of barrier crossing through nonenthalpic processes. We propose a two-group classification of chemical chaperones based upon their thermodynamic effect on RI and TS, which is also supported by single molecule Förster resonance energy transfer (smFRET) studies. Interestingly, for a different test protein, the molecular mechanisms of the two groups of chaperones are not conserved. This provides a glimpse into the complexity of chemical chaperoning activity of osmolytes. Future work would allow us to engineer synergism between the two classes to design more efficient chemical chaperones to ameliorate protein misfolding and aggregation problems.

Hyperthermophile Protein Behavior: Partially-Structured Conformations of Pyrococcus furiosus Rubredoxin Monomers Generated through Forced Cold-Denaturation and Refolding

Some years ago, we showed that thermo-chemically denatured, partially-unfolded forms of Pyrococcus furiosus triosephosphateisomerase (PfuTIM) display cold-denaturation upon cooling, and heat-renaturation upon reheating, in proportion with the extent of initial partial unfolding achieved. This was the first time that cold-denaturation was demonstrated for a hyperthermophile protein, following unlocking of surface salt bridges. Here, we describe the behavior of another hyperthermophile protein, the small, monomeric, 53 residues-long rubredoxin from Pyrococcus furiosus (PfRd), which is one of the most thermostable proteins known to man. Like PfuTIM, PfRd too displays cold-denaturation after initial thermo-chemical perturbation, however, with two differences: (i) PfRd requires considerably higher temperatures as well as higher concentrations of guanidium hydrochloride (Gdm.HCl) than PfuTIM; (ii) PfRd's cold-denaturation behavior during cooling after thermo-chemical perturbation is incompletely reversible, unlike PfuTIM's, which was clearly reversible (from each different conformation generated). Differential cold-denaturation treatments allow PfRd to access multiple partially-unfolded states, each of which is clearly highly kinetically-stable. We refer to these as ‘Trishanku’ unfolding intermediates (or TUIs). Fascinatingly, refolding of TUIs through removal of Gdm.HCl generates multiple partially-refolded, monomeric, kinetically-trapped, non-native ‘Trishanku’ refolding intermediates (or TRIs), which differ from each other and from native PfRd and TUIs, in structural content and susceptibility to proteolysis. We find that the occurrence of cold denaturation and observations of TUI and TRI states is contingent on the oxidation status of iron, with redox agents managing to modulate the molecule's behavior upon gaining access to PfRd's iron atom. Mass spectrometric examination provides no evidence of the formation of disulfide bonds, but other experiments suggest that the oxidation status of iron (and its extent of burial) together determine whether or not PfRd shows cold denaturation, and also whether redox agents are able to modulate its behavior.

Combined Single Molecule Force and Fluorescence Spectroscopy of the Unfolding and Refolding of Green Fluorescent Protein

We have used optical tweezers to study the free energy surface for unfolding-refolding of the green fluorescent protein (EGFP) and simultaneously monitored the loss and recovery of its fluorescence. This conformational landscape shows unfolding intermediates, molten globe refolding intermediates, as well as misfolded states. As force is used to drive transitions between conformational substates, single molecule fluorescence is probed. In its native state, the emission of EGFP is punctuated by transient dark states (blinking); this emission is lost and regained as the protein is unfolded and subsequently refolded. These results provide a full understanding of the unfolding-refolding energy landscape of EGFP and how the conformational state affects the environment of the fluorophore, which reconciles three classes of previous experiments: (1) bulk unfolding/refolding with fluorescence (2) single molecule force-unfolding and (3) single molecule fluorescence intensity fluctuations. This investigation is relevant to efforts at developing EGFP as a genetically encoded force sensor, as well as its use in single molecule imaging and fluorescence recovery after photobleaching experiments.

Functional Intermediate in the Refolding Pathway of a Large and Multidomain Protein Malate Synthase G

Despite their prevalence in biological systems, information about the folding pathways of large and multidomain proteins is meager, as they often unfold irreversibly under in vitro conditions which make their folding studies difficult or even impossible. The folding mechanism of a large (82 kDa) and multidomain protein Malate synthase G (MSG) has been demonstrated in the present study using intrinsic tryptophan fluorescence, enzymatic activity, and extrinsic fluorophore ANS as probes for monitoring the refolding process. Refolding of MSG is found to occur in three kinetic phases. Denatured MSG forms a collapsed state in the burst phase of refolding, which then gives rise to an active intermediate having the same tryptophan fluorescence and enzymatic activity as native MSG in the slow phase. Native topology of MSG is formed from the active intermediate in the very slow phase of refolding which is silent to tryptophan fluorescence change and is susceptible to aggregation at higher protein concentrations. Dependence of rates of very slow phase on GdnHCl concentration suggests that it is not solely a cis/trans proline isomerization limited process but might involve an additional folding event of the domains, not forming the active site of the protein. In light of the above findings, the appearance of a functional intermediate during refolding of MSG was predicted to be an instance of weak interdomain cooperativity. This work has significant implications in the characterization of the refolding intermediates of multidomain proteins in general and MSG in particular, where weak interdomain cooperativity might contribute toward generation of a functional intermediate during its refolding.

Tryptophan dynamics in the exploration of micro-conformational changes of refolded β-lactoglobulin after thermal exposure: A steady state and time-resolved fluorescence approach

Refolding intermediates of proteins, including molten globules, are likely to undergo dynamic conformational transitions. In this work, thermal unfolding and refolding of bovine β-lactoglobulin (β-lg) have been revisited to encounter such intermediate states. Lower thermal range (<80°C) was selected to avoid irreversible aggregate formation. The gross kinetic refolding as monitored with the fluorophore, Trp19, was likely to be reversible but alteration in time resolved fluorescence parameters ruled out the possibility of micro-structural reversibility for the refolded partner. Time resolved fluorescence showed that the refolded protein still lacks some intact native conformation. Far-UV CD signals lack the signature of any secondary structural distortion in global structural context whereas near-UV CD signals were strongly indicative of perturbation in micro-structure surrounding the aromatic moieties which hardly revives after cooling. Steady state anisotropy results showed successfully the break-down of dimer to monomer form of β-lg within 50°C temperature range and augmentation in anisotropy up on further thermal stress reflected the reorganization of tryptophan residues into more restricted and rigid micro-environment as well as irreversible disulfide-linked dimer formation. Reliability of conformational reversibility in the thermal unfolding–refolding is still enigmatic on micro and global structural perspectives. Intermediate state prior to the completion of refolding of thermally exposed β-lg was identified through fluorescence studies.

Varied effects of Pyrococcus furiosus prefoldin and P. furiosus chaperonin on the refolding reactions of substrate proteins

Prefoldin is a molecular chaperone found in the archaeal and eukaryotic cytosol. Prefoldin can stabilize tentatively nascent polypeptide chains or non-native forms of mainly cytoskeletal proteins, which are subsequently delivered to group II chaperonin to accomplish their precise folding. However, the detailed mechanism is not well known, especially with regard to endogenous substrate proteins. Here, we report the effects of Pyrococcus furiosus prefoldin (PfuPFD) on the refolding reactions of Pyrococcus furiosus citrate synthase (PfuCS) and Aequorea enhanced green fluorescence protein (GFPuv) in the presence or absence of Pyrococcus furiosus chaperonin (PfuCPN). We confirmed that both PfuPFD and PfuCPN interacted with PfuCS and GFPuv refolding intermediates. However, the interactions between chaperone and substrate were different for each case, as was the final effect on the refolding reaction. Effects on the refolding reaction varied from passive effects such as ATP-dependent binding and release (PfuCPN towards GFPuv) and binding which leads to folding arrest (PfuPFD towards GFPuv), to active effects such as net increase in thermal stability (PfuCPN towards PfuCS) to an active improvement in refolding yield (PfuPFD towards PfuCS). We postulate that differences in molecular interactions between substrate and chaperone lead to these differences in chaperoning effects.

Slow formation of aggregation‐resistant β‐sheet folding intermediates

Protein folding has been studied extensively for decades, yet our ability to predict how proteins reach their native state from a mechanistic perspective is still rudimentary at best, limiting our understanding of folding-related processes in vivo and our ability to manipulate proteins in vitro. Here, we investigate the in vitro refolding mechanism of a large beta-helix protein, pertactin, which has an extended, elongated shape. At 55 kDa, this single domain, all-beta-sheet protein allows detailed analysis of the formation of beta-sheet structure in larger proteins. Using a combination of fluorescence and far-UV circular dichroism spectroscopy, we show that the pertactin beta-helix refolds remarkably slowly, with multiexponential kinetics. Surprisingly, despite the slow refolding rates, large size, and beta-sheet-rich topology, pertactin refolding is reversible and not complicated by off-pathway aggregation. The slow pertactin refolding rate is not limited by proline isomerization, and 30% of secondary structure formation occurs within the rate-limiting step. Furthermore, site-specific labeling experiments indicate that the beta-helix refolds in a multistep but concerted process involving the entire protein, rather than via initial formation of the stable core substructure observed in equilibrium titrations. Hence pertactin provides a valuable system for studying the refolding properties of larger, beta-sheet-rich proteins, and raises intriguing questions regarding the prevention of aggregation during the prolonged population of partially folded, beta-sheet-rich refolding intermediates. Proteins 2010. (c) 2009 Wiley-Liss, Inc.

Mechanism of pH‐sensitive polymer‐assisted protein refolding and its application in TGF‐β1 and KGF‐2

Refolding of proteins at high concentrations often results in non-productive aggregation. This study, through a unique combination of spectroscopic and chromatographic analyzes, provides biomolecular evidence to demonstrate the ability of Eudragit S-100, a pH-responsive polymer, to enhance refolding of denatured-reduced lysozyme at high concentrations. The addition of Eudragit in the refolding buffer significantly increases lysozyme refolding yield to 75%, when dilution refolding was conducted at 1 mg/mL lysozyme. This study shows evidence of an electrostatic interaction between oppositely charged lysozyme and the Eudragit polymer during refolding. This ionic complexing of Eudragit and lysozyme appears to shield exposed hydrophobic residues of the lysozyme refolding intermediates, thus minimizing hydrophobic-driven aggregation of the molecules. Importantly, results from this study show that the Eudragit-lysozyme bioconjugation does not compromise refolded protein structure, and that the polymer can be readily dissociated from the protein by ion exchange chromatography. The strategy was also applied to refolding of TGF-beta1 and KGF-2.

Molecular-assisted refolding: Study of two different ionic forms of recombinant human fibroblast growth factors

Despite several early reports on the refolding-enhancement effect of a stimuli-responsive polymer, Eudragit, the exact mechanism by which Eudragit interacts with protein molecules to improve refolding remains not well-understood. This study provides clear elucidation of the mode of involvement of Eudragit in facilitating the refolding of two ionic forms of human fibroblast growth factor: (i) human basic fibroblast growth factor (hbFGF) and (ii) human acidic fibroblast growth factor (haFGF). Eudragit enhanced the refolding yield of hbFGF to 74% at 0.5 mg ml −1 refolding protein concentration, but had little effect on its acidic counterpart. Spectroscopic and chromatographic analyses provide clear biomolecular evidence that the oppositely charged polymer and hbFGF protein interact during refolding by forming an ionic complex. This ionic conjugation shields exposed hydrophobic residues of the hbFGF refolding intermediates, thus minimizing hydrophobic-prone aggregation of the molecules. The refolded hbFGF protein can be readily dissociated from the polymer by ion-exchange chromatography. Circular dichroism spectroscopy confirmed that the interaction between Eudragit and hbFGF during refolding did not compromise the final refolded protein structure. This study conclusively shows that Eudragit exhibits charge selectivity in its role as a refolding enhancer, which will have a significant impact on its future use as a refolding additive.

Cleaved Serpin Refolds into the Relaxed State via a Stressed Conformer

Serine proteinase inhibitors (serpins) are believed to fold in vivo into a metastable "stressed" state with cleavage of their P1-P1' bond resulting in reactive center loop insertion and a thermostable "relaxed" state. To understand this unique folding mechanism, we investigated the refolding processes of the P1-P1'-cleaved forms of wild type ovalbumin (cl-OVA) and the R339T mutant (cl-R339T). In the native conditions, cl-OVA is trapped as the stressed conformer, whereas cl-R339T attains the relaxed structure. Under urea denaturing conditions, these cleaved proteins completely dissociated into the heavy (Gly(1)-Ala(352)) and light (Ser(353)-Pro(385)) chains. Upon refolding, the heavy chains of both proteins formed essentially the same initial burst refolding intermediates and then reassociated with the light chain counterparts. The reassociated intermediates both refolded into the native states with indistinguishable kinetics. The two refolded proteins, however, had a notable difference in thermostability. cl-OVA refolded into the stressed form with T(m) = 68.4 degrees C, whereas cl-R339T refolded into the relaxed form with T(m) = 85.5 degrees C. To determine whether cl-R339T refolds directly to the relaxed state or through the stressed state, conformational analyses by anion-exchange chromatography and fluorescence measurements were executed. The results showed that cl-R339T refolds first to the stressed conformation and then undergoes the loop insertion. This is the first demonstration that the P1-P1'-cleaved serpin peptide capable of loop insertion refolds to the stressed conformation. This highlights that the stressed conformation of serpins is an inevitable intermediate state on the folding pathway to the relaxed structure.

Identification of an oxidative refolding intermediate of recombinant consensus interferon from inclusion bodies and design of a two‐stage strategy to promote correct disulfide‐bond formation

Dilution refolding of recombinant consensus IFN (interferon) from inclusion bodies suffers from low yield. A stable intermediate was found to mix with the correct product and to have an antiviral activity of less than 10% of the latter. This intermediate would form precipitates upon removal of the precipitation inhibitor arginine. Compared with the native protein, the intermediate moved more slowly on non-reducing SDS/PAGE. The CD and fluorescence spectra indicated that it had formed a native-like structure, but had only one disulfide bond: Cys(29)-Cys(139). Further evidence showed that the formation of Cys(29)-Cys(139) is specific and very likely to happen, even in the presence of a high concentration of reducing agent, whereas pairing of the other disulfide (Cys(1)-Cys(99)) needed a stronger oxidative condition. It competed with intermolecular disulfide bonding to form covalent oligomers. On the basis of this discovery, a two-stage refolding step strategy was designed that employed a modified dilution refolding step followed by a dialysis refolding step. The first stage used a high concentration of reducing agent together with the precipitation inhibitor arginine. The purpose was to hinder any reaction through Cys(1) or Cys(99) but allow the intramolecular disulfide bonding of Cys(29)-Cys(139). The second stage was a dialysis step that gradually increased the oxidative agent concentration and simultaneously decreased the arginine concentration. The refolding yield was increased from 35 to 82%, while the mass recovery was increased from 60 to 96%. Moreover, this strategy could suppress precipitation even after arginine was completely removed.

Functional Characterization of the Recombinant Group II Chaperonin from Thermoplasma acidophilum

The functional characteristics of group II chaperonins, especially those from archaea, have not been elucidated extensively. Here, we performed a detailed functional characterization of recombinant chaperonin alpha subunits (16-mer) (Ta-cpn alpha) from the thermophilic archaea Thermoplasma acidophilum as a model protein of archaeal group II chaperonins. Recombinant Ta-cpn alpha formed an oligomeric ring structure similar to that of native protein, and displayed an ATP hydrolysis activity (optimal temperature: 60 degrees C) in the presence of either magnesium, manganese or cobalt ions. Ta-cpn alpha was able to bind refolding intermediates of Thermus MDH and GFP in the absence of ATP, and to promote the refolding of Thermus MDH at 50 degrees C in the presence of Mg2+-, Mn2+-, or Co2+-ATP. Ta-cpn alpha also prevented thermal aggregation of rhodanese and luciferase at 50 degrees C. Interestingly, Ta-cpn alpha in the presence of Mn2+ ion showed an increased hydrophobicity, which correlated with an increased efficiency in substrate protein binding. Our finding that Ta-cpn alpha chaperonin system displays folding assistance ability with ATP-dependent substrate release may provide a detailed look at the potential functional capabilities of archaeal chaperonins.

Adsorptive refolding of histidine-tagged glutathione S-transferase using metal affinity chromatography

Column-based protein refolding strategies are often advantageous due to their ease of integration with purification operations, improved refolding yields, and the high concentrations at which the refolded protein can be recovered. His 6-tagged glutathione S-transferase (GST-(His 6)) was refolded while it was adsorbed in a metal affinity chromatography column. The redox environment could be controlled during the refolding reaction by the addition of reduced and oxidized glutathione without reducing the immobilized nickel metal ions. Adsorptive refolding limited the interaction of refolding intermediates at elevated protein concentrations, and thus improved the yield compared to experiments performed using dilution refolding techniques. The protein concentration during refolding was increased by a factor of 6.8 without reducing the yield achieved compared to dilution refolding. The ability of GST-(His 6) to refold to the correct tertiary structure was not significantly affected by the interaction between the poly-histidine-tag and the adsorbent. Decreased refolding yields were achieved at elevated adsorbed protein concentrations, which indicated that at high concentrations the refolding intermediates aggregated despite immobilization. Following adsorptive refolding it was observed that only correctly folded protein could be eluted with imidazole, while the misfolded and aggregated proteins were retained in the column via non-specific interactions with the adsorbent matrix. An iterative refolding strategy was therefore used to re-denature the retained proteins and repeat the adsorptive refolding step, which increased the adsorptive refolding yield that could be achieved at elevated protein concentrations. The yield of correctly folded GST-(His 6) from an iterative refolding process was comparable to dilution refolding performed at a 10-fold lower protein concentration. Selective elution and iterative refolding is likely to improve the yields achieved for other poly-histidine-tagged proteins refolded in metal affinity chromatography columns.

Unfolding of apomyoglobin studied with two-dimensional correlations of tryptophan, 8-anilino-1-naphthalenesulfonate, and pyrene fluorescence

Acid-induced unfolding of horse apomyoglobin is studied with fluorescence of tryptophan residues and extrinsic probes 8-anilino-1-naphthalenesulfonate (ANS) and pyrene. Two-dimensional statistical correlation coefficient analysis of tryptophan, ANS and pyrene fluorescence reveals that apomyoglobin unfolds in two steps via a folding intermediate. Both tryptophan and ANS fluorescence show microenvironment-sensitive emission maximum and fluorescence intensity. Hetero-spectral 2D correlation analysis reveals that the “red” tryptophan fluorescence correlates with the “red” ANS fluorescence, and the “blue” correlates with the “blue” in the acid-induced unfolding process. Correlation curves are established between the hydrophobicity of the probe-binding site(s) as disclosed by ANS and pyrene fluorescence and the folding extent of apomyoglobin as indicated by tryptophan fluorescence. Two anion-induced refolding intermediates of apomyoglobin: Cl −-induced A-1, and trichloroacetate (TCA)-induced I-2 do not follow the correlation trajectory in the acid-induced unfolding. This suggests that the two anion-induced intermediates do not belong to the low-salt acid-unfolding pathway of apomyoglobin.

Effect of Denaturant Concentration on Hen‐egg White Lysozyme Renaturation

AbstractBased on three‐state renaturation process of denatured proteins, an equation describing the effect of denaturant concentration on renaturation yield of denatured proteins was presented. By this equation, two parameters n(m1−m2) and Ka can be obtained. The former indicates the difference in the number of denaturant molecules between the renaturation process of n number of refolding intermediates from refolding intermediate state to native state and their aggregate process from refolding intermediate state to aggregate state, the latter denotes the apparent aggregate equilibrium constant for protein molecules aggregated from native state to aggregate state, and from them, the characteristics of the renaturation process of denatured proteins in denaturant solution can be identified. This equation was tested by the renaturation processes of denatured egg white lysozyme in guanidine hydrochloride and urea solutions, with the results to show that when guanidine hydrochloride and urea concentrations were separately higher than 1.25 and 3.00 mol/L or separately lower than 1.00 and 3.00 mol/L, the refolding intermediates of egg white lysozymes were more easily aggregated to aggregate state or more easily renatured to native state, respectively. Under different initial total egg white lysozyme concentrations in urea solution, the refolding egg white lysozyme intermediates could be deduced to have a tendency to form a bimolecular intermediate aggregate, and this inference was further confirmed by their nonreducing SDS‐PAGE and size exclusion chromatography.