Structural characterization of apomyoglobin self-associated species in aqueous buffer and urea solution

The biophysical characterization of nonfunctional protein aggregates at physiologically relevant temperatures is much needed to gain deeper insights into the kinetic and thermodynamic relationships between protein folding and misfolding. Dynamic and static laser light scattering have been employed for the detection and detailed characterization of apomyoglobin (apoMb) soluble aggregates populated at room temperature upon dissolving the purified protein in buffer at pH 6.0, both in the presence and absence of high concentrations of urea. Unlike the beta-sheet self-associated aggregates previously reported for this protein at high temperatures, the soluble aggregates detected here have either alpha-helical or random coil secondary structure, depending on solvent and solution conditions. Hydrodynamic diameters range from 80 to 130 nm, with semiflexible chain-like morphology. The combined use of low pH and high urea concentration leads to structural unfolding and complete elimination of the large aggregates. Even upon starting from this virtually monomeric unfolded state, however, protein refolding leads to the formation of severely self-associated species with native-like secondary structure. Under these conditions, kinetic apoMb refolding proceeds via two parallel routes: one leading to native monomer, and the other leading to a misfolded and heavily self-associated state bearing native-like secondary structure.     

Hexafluoroacetone hydrate as a structure modifier in proteins: characterization of a molten globule state of hen egg-white lysozyme.

A molten globule-like state of hen egg-white lysozyme has been characterized in 25% aqueous hexafluoroacetone hydrate (HFA) by CD, fluorescence, NMR, and H/D exchange experiments. The far UV CD spectra of lysozyme in 25% HFA supports retention of native-like secondary structure while the loss of near UV CD bands are indicative of the overall collapse of the tertiary structure. The intermediate state in 25% HFA exhibits an enhanced affinity towards the hydrophobic dye, ANS, and a native-like tryptophan fluorescence quenching. 1-D NMR spectra indicates loss of native-like tertiary fold as evident from the absence of ring current-shifted 1H resonances. CD, fluorescence, and NMR suggest that the transition from the native state to a molten globule state in 25% HFA is a cooperative process. A second structural transition from this compact molten globule-like state to an "open" helical state is observed at higher concentrations of HFA (> or = 50%). This transition is characterized by a dramatic loss of ANS binding with a concomitant increase in far UV CD bands. The thermal unfolding of the molten globule state in 25% HFA is sharply cooperative, indicating a predominant role of side-chain-side-chain interactions in the stability of the partially folded state. H/D exchange experiments yield higher protection factors for many of the backbone amide protons from the four alpha-helices along with the C-terminal 3(10) helix, whereas little or no protection is observed for most of the amide protons from the triple-stranded antiparallel beta-sheet domain. This equilibrium molten globule-like state of lysozyme in 25% HFA is remarkably similar to the molten globule state observed for alpha-lactalbumin and also with the molten globule state transiently observed in the kinetic refolding experiments of hen lysozyme. These results suggest that HFA may prove generally useful as a structure modifier in proteins.     

Modeling and simulation of fed-batch protein refolding process

The simplified kinetic model that assumes competition between first-order folding and third-order aggregation was used to model the fed-batch refolding of denatured-reduced lysozyme. It was found that the model was able to describe the process at limited concentration ranges, i.e., 1-2 and 5-7 mg mL(-)(1), respectively, at extensive guanidinium chloride (GdmCl) concentrations and controlled concentrations of oxidizing and reducing agents. The folding or aggregation rate constant was different at the two protein concentration ranges and strongly dependent on the denaturant concentration. As a result, both rate constants at the two concentration ranges were expressed as functions of GdmCl concentration. The rate constants determined by fed-batch experiments could be employed for the prediction of the fed-batch process but were not able to be extended to a batch refolding by direct dilution. Computer simulations show that the denaturant concentration and fed-batch flow rate are important factors influencing the refolding yield. Prolonged fed-batch time is beneficial to keep the transient intermediate concentration at a low level and to increase the yield of correctly folded protein. This is of importance when the denaturant concentration in refolding buffer solution is low. Thus, at a low denaturant concentration, fed-batch time should be sufficiently long, whereas at an appropriately high GdmCl concentration, a short fed-batch time or a high feed rate of the denatured protein is effective to give a high refolding yield.     

Multiple-probe analysis of folding and unfolding pathways of human serum albumin. Evidence for a framework mechanism of folding.

The changes in the far-UV CD signal, intrinsic tryptophan fluorescence and bilirubin absorbance showed that the guanidine hydrochloride (GdnHCl)-induced unfolding of a multidomain protein, human serum albumin (HSA), followed a two-state process. However, using environment sensitive Nile red fluorescence, the unfolding and folding pathways of HSA were found to follow a three-state process and an intermediate was detected in the range 0.25-1.5 m GdnHCl. The intermediate state displayed 45% higher fluorescence intensity than that of the native state. The increase in the Nile red fluorescence was found to be due to an increase in the quantum yield of the HSA-bound Nile red. Low concentrations of GdnHCl neither altered the binding affinity of Nile red to HSA nor induced the aggregation of HSA. In addition, the secondary structure of HSA was not perturbed during the first unfolding transition (<1.5 m GdnHCl); however, the secondary structure was completely lost during the second transition. The data together showed that the half maximal loss of the tertiary structure occurred at a lower GdnHCl concentration than the loss of the secondary structure. Further kinetic studies of the refolding process of HSA using multiple spectroscopic techniques showed that the folding occurred in two phases, a burst phase followed by a slow phase. An intermediate with native-like secondary structure but only a partial tertiary structure was found to form in the burst phase of refolding. Then, the intermediate slowly folded into the native state. An analysis of the refolding data suggested that the folding of HSA could be best explained by the framework model.     

Structural characterization of acid-induced intermediates of human glutathione transferase P1-1

The acid denaturation of human glutathione transferase P1-1 (hGSTP1-1) has been performed to investigate the unfolding intermediates of the protein and their possible involvement in the refolding mechanism. The acid-induced structures of GSTP1-1 have been characterized by activity, gel filtration, intrinsic fluorescence and far-u.v. circular dichroism (CD) techniques. Because of the non-identity of the different transitions monitored, the acid denaturation of hGSTP1-1 appears to be a multistep process during which several intermediates coexist in equilibrium. The dependence of inactivation on the protein concentration, as well as gel-filtration experiments, indicate that the inactivation transition, centred at about pH 4.0, corresponds to the monomerization of the protein. At pH 2.0, when the enzyme is completely inactive, the protein retains a small, but significant, amount of secondary structure. This means that the dimeric arrangement of the molecule is important for maintaining the native-like secondary structure of the monomer. The results show that, at low pH, the compact state of the GST monomer, even upon the addition of salts, does not possess native-like secondary structure as described for many monomeric proteins (molten globule). In the presence of physiological concentrations of salts, the protein solution at pH 2.0 leads to a dead-end aggregation process, suggesting that this compact state cannot represent a productive intermediate of the refolding pathway.     

Intestinal fatty acid binding protein: the folding mechanism as determined by NMR studies

The intestinal fatty acid binding protein is composed of two beta-sheets surrounding a large interior cavity. There is a small helical domain associated with the portal for entry of the ligand into the cavity. Denaturation of the protein has been monitored in a residue-specific manner by collecting a series of two-dimensional (1)H-(15)N heteronuclear single-quantum coherence (HSQC) NMR spectra from 0 to 6.5 M urea under equilibrium conditions. In addition, rates for hydrogen-deuterium exchange have been measured as a function of denaturant concentration. Residual, native-like structure persists around hydrophobic clusters at very high urea concentrations. This residual structure (reflecting only about 2-7% persistence of native-like structure) involves the turns between beta-strands and between the two short helices. If this persistence is assumed to reflect transient native-like structure in these regions of the polypeptide chain, these sites may serve as nucleation sites for folding. The data obtained at different urea concentrations are then analyzed on the basis of peak intensities relative to the intensities in the absence of urea reflecting the extent of secondary structure formation. At urea concentrations somewhat below 6.5 M, specific hydrophobic residues in the C-terminal beta-sheet interact and two strands, the D and E strands in the N-terminal beta-sheet, are stabilized. These latter strands surround one of the turns showing residual structure. With decreasing urea concentrations, the remaining strands are stabilized in a specific order. The early strand stabilization appears to trigger the formation of the remainder of the C-terminal beta-sheet. At low urea concentrations, hydrogen bonds are formed. A pathway is proposed on the basis of the data describing the early, intermediate, and late folding steps for this almost all beta-sheet protein. The data also show that there are regions of the protein which appear to act in a concerted manner at intermediate steps in refolding.     

A Partially Folded State of Ovalbumin at Low pH Tends to Aggregate

At pH 2, ovalbumin retains native-like secondary structure as seen by far-UV CD and FTIR, but lacks well-defined tertiary structure as seen by the fluorescence and near-UV CD spectra. Addition of 20 mM Trifluoroacetic acid (TFA) or 30 mM Trichloroacetic acid (TCA) on acid-induced state results in protein aggregation. This aggregated state possesses extensive β-sheet structure as revealed by far-UV CD and FTIR spectroscopy. Furthermore, the aggregates exhibit decreased ANS fluorescence and increased thioflavin T fluorescence. The presence of aggregates was confirmed by size exclusion chromatography. Such a formation of β-sheet structure is found in the amyloid of a number of neurological diseases such as Alzheimer’s and scrapie. Ovalbumin at low pH, in the presence of K₂SO₄, exists in partially folded state characterized by native-like secondary structure and tertiary folds.     

Critical analysis of lysozyme refolding kinetics

The kinetics of lysozyme refolding and aggregation is studied using an existing competing first- and third-order reaction scheme. The existing model overestimates yield at high refolding concentrations (>1 mg/mL), thus limiting its use for reactor design at industrially relevant refolding concentrations. This study demonstrates that a pathway exists for the incorporation of refolded native protein into aggregates. Specifically, native lysozyme labeled with fluorescein isothiocyanate was added to the refolding buffer prior to dilution refolding of denatured and reduced lysozyme. Aggregates collected from these experiments showed significant fluorescence, indicating that labeled lysozyme had been incorporated into the aggregates during refolding. Although the precise pathway of incorporation has not been elucidated, it is clear from this work that the existing model for lysozyme refolding is not globally applicable. In particular, previous work has analytically demonstrated that neglect of a pathway from native to aggregate can result in the design of a grossly suboptimal reactor strategy. This study demonstrates that such a pathway can exist experimentally and emphasizes the need to critically assess refolding kinetic models before their use in reactor design equations.     

A kinetic study of the competition between renaturation and aggregation during the refolding of denatured-reduced egg white lysozyme

The recovery of proteins following denaturation is optimal at low protein concentrations. The decrease in yield at high concentrations has been explained by the kinetic competition of folding and "wrong aggregation". In the present study, the renaturation-reoxidation of hen and turkey egg white lysozyme was used as a model system to analyze the committed step in aggregate formation. The yield of renatured protein for both enzymes decreased with increasing concentration in the folding process. In addition, the yield decreased with increasing concentrations of the enzyme in the denatured state (i.e., prior to its dilution in the renaturation buffer). The kinetics of renaturation of turkey lysozyme were shown to be very similar to those of hen lysozyme, with a half-time of about 4.5 min at 20 degrees C. The rate of formation of molecular species that lead to formation of aggregates (and therefore fail to renature) was shown to be rapid. Most of the reaction occurred in less than 5 s after the transfer to renaturation buffer, and after 1 min, the reaction was essentially completed. Yet, by observing the effects of the delayed addition of denatured hen lysozyme to refolding turkey lysozyme, it was shown that folding intermediates become resistant to aggregation only much more slowly, with kinetics indistinguishable from those observed for the appearance of native molecules. The interactions leading to the formation of aggregates were nonspecific and do not involve disulfide bonds. These observations are discussed in terms of possible kinetic and structural aspects of the folding pathway.     

Structure of folding intermediates at pH 4.0 and native state of microbial transglutaminase

Recombinant microbial transglutaminase has been expressed in Escherichia coli as insoluble inclusion bodies. After we searched for refolding conditions, refolding of the protein could be done by first dilution of the unfolded enzyme in a buffer at pH 4.0, and then by titration of the pH from 4.0 to 6.0. CD analysis showed that a burst of secondary structure formation occurred within the dead time of the experiment and accounted for 75% of the signal change in the far UV CD, with little tertiary structure being formed. This burst was followed by slow rearrangement of the secondary structure accompanied by formation of tertiary structure. The secondary and tertiary structures of the final sample at pH 4.0, corresponding to the folding intermediate, were different from these structures at pH 6.0. Once the native structure was obtained, acidification of the native protein to pH 4.0 did not lead to a structure like that of the folding intermediate. Sedimentation velocity analysis showed that the folding intermediate had an expanded structure and contained no other structure species including large aggregates.     

beta-sheet structure formation of proteins in solid state as revealed by circular dichroism spectroscopy

Cross beta-sheet structure formation and abnormal aggregation of proteins are thought to be pathological characteristics of some neurodegenerative disorders. To investigate the novel structural transformation and aggregation, the solid-state secondary structures of some proteins and peptides associated in thin films were determined by circular dichroism spectroscopy. Insulin, lysozyme, DsbA protein, luciferase, and ovalbumin peptide fall into one group; they show no or slight structural rearrangement from solution to the solid state. Another group, including bovine serum albumin, ovalbumin, alpha-synuclein, and plasminogen activator inhibitor-1 (PAIRC) peptide, undergo structural transformation with an increase of beta-sheet structure in the solid state. The beta-sheet formation of PAIRC peptide may reflect the structural transformation of the serpin reactive center that is relevant to the inhibitor activity. The beta-sheet structure of alpha-synuclein in the solid state may correspond to the amyloid-like aggregates, which are implicated in the pathogenesis of some neurodegenerative diseases.     

The oxidative refolding of hen lysozyme and its catalysis by protein disulfide isomerase.

The oxidative refolding of hen lysozyme has been studied by a variety of time-resolved biophysical methods in conjunction with analysis of folding intermediates using reverse-phase HPLC. In order to achieve this, refolding conditions were designed to reduce aggregation during the early stages of the folding reaction. A complex ensemble of relatively unstructured intermediates with on average two disulfide bonds is formed rapidly from the fully reduced protein after initiation of folding. Following structural collapse, the majority of molecules slowly form the four-disulfide-containing fully native protein via rearrangement of a highly native-like, kinetically trapped intermediate, des-[76-94], although a significant population (approximately 30%) appears to fold more quickly via other three-disulfide intermediates. The folding catalyst PDI increases dramatically both yields and rates of lysozyme refolding, largely by facilitating the conversion of des-[76-94] to the native state. This suggests that acceleration of the folding rate may be an important factor in avoiding aggregation in the intracellular environment.     

Refolding, structural transition and spermatozoa-binding of recombinant bonnet monkey (Macaca radiata) zona pellucida glycoprotein-C expressed in Escherichia coli.

An internal cDNA fragment (978 bp) corresponding to bonnet monkey (Macaca radiata) zona pellucida glycoprotein-C (bmZPC), excluding the N-terminal signal sequence and the C-terminal transmembrane-like domain, was cloned in pQE-30 vector and the protein expressed as inclusion bodies in Escherichia coli. Recombinant bmZPC (r-bmZPC) was solubilized from purified inclusion bodies in the absence of a high concentration of chaotropic agents and was subsequently refolded. Use of a low concentration of urea (2 M) during solubilization of r-bmZPC helped to minimize the extent of protein aggregation during refolding of the recombinant protein, and retain the existing native-like secondary structure that was essential for proper folding. Purified r-bmZPC appeared as a dominant band of 43 kDa on SDS/PAGE and Western blot. Although it lacked carbohydrate moieties, the purified and refolded r-bmZPC bound to the head region of bonnet monkey spermatozoa, confirming the existence of a native-like conformation. CD revealed a maximum at 200 nm and a single broad minimum extending from 209 to 216 nm, indicating the presence of both alpha-helical and beta-sheet conformations in the refolded r-bmZPC. Two different phases of transition were observed by urea-gradient electrophoresis, suggesting the existence of multiple intermediate stages during the unfolding of r-bmZPC. The availability of refolded r-bmZPC will help in elucidating its role during the complex cascade of events during fertilization.     

Identification of equilibrium and kinetic intermediates involved in folding of urea-denatured creatine kinase

The unfolding transition and kinetic refolding of dimeric creatine kinase after urea denaturation were monitored by intrinsic fluorescence and far ultraviolet circular dichroism. An equilibrium intermediate and a kinetic folding intermediate were identified and characterized. The fluorescence intensity of the equilibrium intermediate is close to that of the unfolded state, whereas its ellipticity at 222 nm is about 50% of the native state. The transition curves measured by these two methods are therefore non-coincident. The kinetic folding intermediate, formed during the burst phase of refolding under native-like conditions, possesses 75% of the native secondary structure, but is mostly lacking in native tertiary structure. In moderate concentrations of urea, only the initial, rapid change in fluorescence intensity or negative ellipticity is observed, and the final state values do not reach the equivalent unfolding values. The unfolding and refolding transition curves measured under identical conditions are non-coincident within the transition from intermediate to fully unfolded state. It is observed by SDS-PAGE that disulfide bond-linked dimeric or oligomeric intermediates are formed in moderate urea concentrations, especially in the refolding reaction. These rapidly formed, soluble intermediates represent an off-pathway event that leads to the hysteresis in the refolding transition curves.     

In Vitro Hyperglycemic Condition Facilitated the Aggregation of Lysozyme via the Passage Through a Molten Globule State

Hyperglycemic condition i.e. an increase in blood glucose concentration has been linked to bring about structural alterations in the native state of proteins. Glucose concentrations of 50 and 100 mM in vitro, which correspond to hyperglycemic condition, were tested to investigate their effect on lysozyme native structure. Incubating enzyme with 50 and 100 mM glucose for a period of 7 days, an intermediate state on day 4 and 3 was observed, respectively. The presence of intermediate state was characterized by a 22 % increase in the intrinsic fluorescence intensity with a red shift of 20 nm compared to the native state, a 5 % increase in ANS-fluorescence intensity relative to the native due to the surfacing of hydrophobic clusters and a sharp decrease in near-UV CD signal at around 284 and 291 nm. The state retains substantial native-like secondary structure. This partially unfolded intermediate state can be referred as ‘molten globule’, which finally tends to aggregate on day 6 and 4 with 50 and 100 mM glucose concentration, respectively, as a result of cross-linking between lysozyme molecules. The aggregates were confirmed by the presence of β-sheet structure as depicted by far-UV CD, an increase in ThT fluorescence as well as the fibrillar morphology shown by SEM. Moreover, advanced glycation end products were also accompanied as the emission peak was observed at 460 and 470 nm corresponding to the formation of pentosidine and malonaldehyde, respectively.     

Chaperone characteristics of PDI-related protein A from Aspergillus niger

The functional properties of a novel protein, protein disulfide isomerase-related protein A (PRPA) from Aspergillus niger T21, have been characterized. (1) PRPA possesses disulfide isomerase activity. (2) In Hepes buffer, at substoichiometric concentrations, PRPA facilitates the formation of inactive lysozyme aggregates associated with PRPA (anti-chaperone activity); while at a high molar excess, PRPA inhibits aggregation by maintaining lysozyme in a soluble, yet inactive, state (chaperone-like activity). However, PRPA only exhibits chaperone-like activity during lysozyme refolding in phosphate buffer. (3) Experiments have indicated that disulfide cross-linkage is not required for the interaction between PRPA and lysozyme, and hydrophobic interaction may be responsible for PRPA effect on lysozyme. (4) Co-expression of PRPA and prochymosin in Escherichia coli leads to reduction of inclusion bodies, rendering part of prochymosin molecules soluble yet inactive. The structural and functional characteristics of PRPA suggest that PRPA may play an important role in protein folding, aggregation, and retention in the endoplasmic reticulum.     

Conformational features of reduced and disulfide intact forms of hen egg white lysozyme in aqueous solution in presence of 3-chloro-1, 2-propanediol and dioxane: implications for protein folding intermediates

Conformational features of reduced and disulfide intact hen egg white lysozyme in aqueous 1,4-dioxane and 3-chloro-1, 2-propanediol solutions have been examined using circular dichroism and fluorescence spectroscopy. We find that in presence of 1, 4-dioxane, reduced lysozyme assumes a relatively compact conformational form with secondary structure closer to native state and no tertiary structure as judged by peptide and aromatic CD spectra and ANS binding studies monitored by fluorescence. Further, in presence of 40% (v/v) 3-chloro-1, 2-propanediol, disulfide intact lysozyme (DI-lysozyme) assumes a conformational form with native like secondary structure and no tertiary structure akin to a molten globule state. We correlate our results to kinetic hydrogen- deuterium exchange NMR results of the refolding of lysozyme available in literature and suggest that the conformational forms observed in our study could be models for kinetic intermediates in the refolding of lysozyme.     

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‐β1 and KGF‐2. © 2009 American Institute of Chemical Engineers Biotechnol. Prog. 2009     

Detection and characterization of an early folding intermediate of T4 lysozyme using pulsed hydrogen exchange and two-dimensional NMR.

Two-dimensional 1H-15N NMR techniques combined with pulsed hydrogen-deuterium exchange have been used to characterize the folding pathway of T4 lysozyme. In the unfolded state, there is little differential protection of the various amides from hydrogen exchange. In the native folded structure, 84 amides of the 164 residues are sufficiently spectrally resolved and protected from solvent exchange to serve as probes of the folding pathway. These probes are located in both the N-terminal and C-terminal domains of the native folded structure of the protein. The studies described here show that at least one intermediate is formed early during refolding at low denaturant concentrations. This intermediate (or intermediates) forms very rapidly (within the 10-ms temporal resolution of our mixing device) under the conditions used and is completed at least 10 times faster than the overall folding event. The intermediate(s) protect(s) from exchange a subset of amides in the N-terminal and C-terminal regions of the protein. In the final folded states these protected regions correspond to two alpha-helices and a beta-sheet region. These amides are protected from exchange by factors between 20 and 200 as compared to the fully unfolded protein. Protection of this magnitude is consistent with the formation of somewhat exposed secondary structure in these regions and could represent a "molten globule"-like or a "framework"-like structure for the intermediate(s) in which specific parts of the sequence form isolated secondary structures that are not stabilized by extensive tertiary interactions.     

An early immunoreactive folding intermediate of the tryptophan synthease beta 2 subunit is a 'molten globule'.

The refolding kinetics of the tryptophan synthase beta 2 subunit have been investigated by circular dichroism (CD) and binding of a fluorescent hydrophobic probe (ANS), using the stopped-flow technique. The kinetics of regain of the native far UV CD signal show that, upon refolding of urea denatured beta 2, more than half of the protein secondary structure is formed within the dead time of the CD stopped-flow apparatus (0.013 s). On the other hand, upon refolding of guanidine unfolded beta 2, the fluorescence of ANS passes through a maximum after about 1 s and then 'slowly' decreases. These results show the accumulation, in the 1-10 s time range, of an early transient folding intermediate which has a pronounced secondary structure and a high affinity for ANS. In this time range, the near UV CD remains very low. This transient intermediate thus appears to have all the characteristics of the 'molten globule' state [(1987) FEBS Lett. 224, 9-13]. Moreover, by comparing the intrinsic time of the disappearance of this transient intermediate (t1/2 35 s) with the time of formation of the previously characterized [(1988) Biochemistry 27, 7633-7640] early immunoreactive intermediate recognized by a monoclonal antibody (t1/2 12 s), it is shown that this native-like epitope forms within the 'molten globule', before the tight packing of the protein side chains.