Effects of proline mutations on the unfolding and refolding of human lysozyme: the slow refolding kinetic phase does not result from proline cis-trans isomerization

The unfolding and refolding kinetics of six proline mutants of the human lysozyme (h-lysozyme) were carried out and compared to that of the wild-type protein. Our results show that the slow refolding phase observed in the h-lysozyme refolding kinetics cannot be ascribed to proline isomerization reactions. The h-lysozyme contains two proline residues at positions 71 and 103, both in the trans conformation in the native state. The refolding kinetics of the P71G/P103G mutant, in which both prolines have been replaced by a glycine, were found to be similar to those of the wild-type protein. The same slow phase amplitude of about 10% was found for both proteins, and the slow phase rate constants were also identical within experimental error. Other mutants such as P103G or P71G, in which only one of the two prolines has been replaced by a glycine, and A47P with its three prolines, gave identical slow refolding phases. The X-ray structure analysis and scanning microcalorimetric study of each protein (Herning et al., unpublished experiments) have confirmed that none of the considered mutations affects significantly protein structure and that no major changes in protein stability were brought about by these mutations. Therefore, comparison of the properties of the mutant and wild-type proteins is legitimate. Interestingly, the refolding kinetics of the V110P mutant, in which a proline residue has been introduced at position 110 (N-terminus of an alpha-helix), were clearly triphasic. For this mutant an additional very slow phase with properties similar to those expected from the proline hypothesis was detected. Equilibrium denaturation studies were conducted for each protein, and the refolding pathway of h-lysozyme is partly presented. We also discuss the effect of proline mutations on the energetics of the folding pathway of the h-lysozyme in water.     

Refolding of the immunoglobulin light chain.

The kinetics of the refolding reactions of type lambda Bence Jones proteins from 4 M GuHCl were studied by CD, ultraviolet absorption, and fluorescence spectrophotometry. The kinetics were complex and consisted of at least three phases, an undetectable fast phase, a detectable fast phase, and a slow phase. The slow phase followed first-order kinetics and the three experimental methods used gave similar rate constants for all the Bence Jones proteins (about 3 X 10(-3) s-1). The refolding reaction of VL fragment was too fast to be measured in the present experiments. The refolding process of CL fragment was very similar to those of Bence Jones proteins except that the detectable fast phase was less significant. The rate constant of the slow phase observed for the CL fragment was similar to those of the slow phase observed for Bence Jones proteins. The activation energy of the slow phase was the same for a Bence Jones protein and its CL fragment. These results indicate that the refolding kinetics of the CL domain are very similar to those of isolated CL fragment and that refolding of the VL domain precedes refolding of the CL domain, even though both domains have similar immunoglobulin folds. However, the results of refolding experiments on Bence Jones proteins, and VL and CL fragments in the presence of ANS, as well as the other lines of evidence, indicate that the refolding kinetics of the Bence Jones protein molecule cannot be expressed as simple sum of the refolding reactions of isolated VL and CL fragments.     

Secretory ribonuclease genes and pseudogenes in true ruminants

Mammalian pancreatic ribonucleases (RNase) form a family of extensively studied homologous proteins. Phylogenetic analyses, based on the primary structures of these enzymes, indicated that the presence of three homologous enzymes (pancreatic, seminal and brain ribonucleases) in the bovine species is due to gene duplication events, which occurred during the evolution of ancestral ruminants. In this paper the sequences are reported of the coding regions of the orthologues of the three bovine secretory ribonucleases in hog deer and roe deer, two deer species belonging to two different subfamilies of the family Cervidae. The sequences of the 3′ untranslated regions of the three different secretory RNase genes of these two deer species and giraffe are also presented. Comparison of these and previously determined sequences of ruminant ribonucleases showed that the brain-type enzymes of giraffe and these deer species exhibit variations in their C-terminal extensions. The seminal-type genes of giraffe, hog deer and roe deer show all the features of pseudogenes. Phylogenetic analyses, based on the complete coding regions and parts of the 3′ untranslated regions of the three different secretory ribonuclease genes of ox, sheep, giraffe and the two deer species, show that pancreatic, seminal- and brain-type RNases form three separate groups.     

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 β‐helix protein, pertactin, which has an extended, elongated shape. At 55 kDa, this single domain, all‐β‐sheet protein allows detailed analysis of the formation of β‐sheet structure in larger proteins. Using a combination of fluorescence and far‐UV circular dichroism spectroscopy, we show that the pertactin β‐helix refolds remarkably slowly, with multiexponential kinetics. Surprisingly, despite the slow refolding rates, large size, and β‐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 β‐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, β‐sheet‐rich proteins, and raises intriguing questions regarding the prevention of aggregation during the prolonged population of partially folded, β‐sheet‐rich refolding intermediates. Proteins 2010. © 2009 Wiley‐Liss, Inc.     

Refolding intermediate of guanidine hydrochloride denatured aminoacylase

The refolding of aminoacylase denatured in 6M guanidine hydrochloride (GdnHCl) has been studied by measuring enzyme activity, fluorescence emission spectra, ANS fluorescence spectra and far-UV circular dichroism spectra. The results showed that GdnHCl-denatured aminoacylase could be refolded and reactivated by dilution. A refolding intermediate was observed for low concentrations of GdnHCl (between 0.5 and 1.2M). This refolding intermediate was characterized by an increased fluorescence emission intensity, a blue-shifted emission maximum, and by increased binding of the fluorescence probe 8-anilino-1-naphthalenesulfonate (ANS). The secondary structure of the intermediate was similar to that of the native enzyme, and was therefore quite similar to the molten globule state often found in the protein folding pathway. Combined with the previous evidence of existence of an intermediate during unfolding process, we therefore proposed that the unfolding and refolding of aminoacylase might share the same pathway. A comparison of the Apo-enzyme and Holo-enzyme showed that there was little effect of the zinc ion on the refolding of the aminoacylase. Our study, the first successful report of the refolding of this metalloenzyme, also showed that lowering the concentration and the temperature of the enzyme improved the refolding rate of aminoacylase. The system therefore provides a useful model to study the refolding of proteins with prosthetic groups.     

Folding kinetics of the all-beta-sheet protein human basic fibroblast growth factor, a structural homolog of interleukin-1beta

The refolding and unfolding kinetics of the all-beta-sheet protein human basic fibroblast growth factor (hFGF-2) were studied by fluorescence spectroscopy. The kinetics of the unfolding transition are monophasic. The refolding reaction at high and low guanidinium chloride (GdmCl) concentrations is best described by mono- and biphasic folding, respectively. Refolding and unfolding of hFGF-2 (155 amino acids) is very slow compared with other non-disulfide-bonded monomeric proteins of similar size. For example, the rate constant for unfolding at 4.5 mol.liter(-1) GdmCl is 0.006 s(-1), and the refolding rate constants at 0.4 mol.liter(-1) GdmCl are 0.01 s(-1) and 0.0009 s(-1) (15 degrees C, pH 7.0). A characterization of the thermodynamic nature of the folding process using transition state theory revealed that the slow refolding is almost exclusively controlled by entropic factors, namely the strong loss of conformational freedom during refolding. The rate of the slow unfolding kinetics is mainly (and at low denaturant concentrations exclusively) controlled by the large positive change in enthalpy. hFGF-2 shows similar slow folding kinetics to that of its structural homolog interleukin-1beta. Since both proteins show very little sequence identity, it is suggested that their slow folding kinetics are determined by the complex beta-sheet arrangement of the native molecules.     

Unusually slow denaturation and refolding processes of pyrrolidone carboxyl peptidase from a hyperthermophile are highly cooperative: real-time NMR studies

The refolding rate of heat-denatured cysteine-free pyrrolidone carboxyl peptidase (PCP-0SH) from Pyrococcus furiosus has been reported to be unusually slow under some conditions. To elucidate the structural basis of the unusually slow kinetics of the protein, the denaturation and refolding processes of the PCP-0SH were investigated using a real-time 2D (1)H-(15)N HSQC and CD experiments. At 2 M urea denaturation of the PCP-0SH in the acidic region, all of the native peaks in the 2D HSQC spectrum completely disappeared. The conformation of the PCP-0SH just after removal of 6 M GuHCl could be observed as a stable intermediate (D(1) state) in 2D HSQC and CD experiments, which is similar to a molten globule structure. The D(1) state of the PCP-0SH, which is the initial state of refolding, corresponded to the state at 2 M urea and seemed to be the denatured state in equilibrium with the native state under the physiological conditions. The refolding of PCP-0SH from the D(1) state to the native state could be observed to be highly cooperative without any intermediates between them, even if the refolding rate was quite slow. In the higher concentration of denaturants, PCP-0SH showed HSQC and CD spectra characteristic of completely unfolded proteins called the D(2) state. The unusually slow refolding rate was discussed as originating in the conformations in the transition state and/or the retardation of reorganization in an ensemble of nonrandom denatured structures in the D(1) state.     

A near-native state on the slow refolding pathway of hen lysozyme

The refolding of four disulfide lysozyme (at pH 5.2, 20 degrees C) involves parallel pathways, which have been proposed to merge at a near-native state. This species contains stable structure in the alpha- and beta-domains but lacks a functional active site. Although previous experiments have demonstrated that the near-native state is populated on the fast refolding pathway, its relevance to slow refolding molecules could not be directly determined from previous experiments. In this paper, we describe experiments that investigate the effect of added salts on the refolding pathway of lysozyme at pH 5.2, 20 degrees C. We show, using stopped flow tryptophan fluorescence, inhibitor binding, and circular dichroism (CD), that the rate of formation of native lysozyme on the slow refolding track is significantly reduced in solutions of high ionic strength in a manner dependent on the position of the anion in the Hofmeister series. By contrast, the rate of evolution of hydrogen exchange (HX) protection monitored by electrospray ionization mass spectrometry (ESI MS) is unchanged under the refolding conditions studied. The data show, therefore, that at high ionic strengths beta-domain stabilization and native state formation on the slow refolding pathway become kinetically decoupled such that the near-native state becomes significantly populated. Thus, by changing the energy landscape with the addition of salts new insights into the relevance of intermediate states in lysozyme refolding are revealed.     

Allelic polymorphism in Arabian camel ribonuclease and the amino acid sequence of bactrian camel ribonuclease

Pancreatic ribonucleases from several species (whitetail deer, roe deer, guinea pig, and arabian camel) exhibit more than one amino acid at particular positions in their amino acid sequences. Since these enzymes were isolated from pooled pancreas, the origin of this heterogeneity is not clear. The pancreatic ribonucleases from 11 individual arabian camels (Camelus dromedarius) have been investigated with respect to the lysine-glutamine heterogeneity at position 103 (Welling et al., 1975). Six ribonucleases showed only one basic band and five showed two bands after polyacrylamide gel electrophoresis, suggesting a gene frequency of about 0.75 for the Lys gene and about 0.25 for the Gln gene. The amino acid sequence of bactrian camel (Camelus bactrianus) ribonuclease isolated from individual pancreatic tissue was determined and compared with that of arabian camel ribonuclease. The only difference was observed at position 103. In the ribonucleases from two unrelated bactrian camels, only glutamine was observed at that position.Part of this work has been carried out under the auspices of the Netherlands Foundation for Chemical Research (S.O.N.) and with financial aid from the Netherlands Organisation for the Advancement of Pure Research (Z.W.O.).     

Unassisted refolding of urea unfolded rhodanese

In vitro refolding after urea unfolding of the enzyme rhodanese (thiosulfate:cyanide sulfurtransferase, EC 2.8.1.1) normally requires the assistance of detergents or chaperonin proteins. No efficient, unassisted, reversible unfolding/folding transition has been demonstrated to date. The detergents or the chaperonin proteins have been proposed to stabilize folding intermediates that kinetically limit folding by aggregating. Based on this hypothesis, we have investigated a number of experimental conditions and have developed a protocol for refolding, without assistants, that gives evidence of a reversible unfolding transition and leads to greater than 80% recovery of native enzyme. In addition to low protein concentration (10 micrograms/ml), low temperatures are required to maximize refolding. Otherwise optimal conditions give less than 10% refolding at 37 degrees C, whereas at 10 degrees C the recovery approaches 80%. The unfolding/refolding phases of the transition curves are most similar in the region of the transition, and refolding yields are significantly reduced when unfolded rhodanese is diluted to low urea concentrations, rather than to concentrations near the transition region. This is consistent with the formation of "sticky" intermediates that can remain soluble close to the transition region. Apparently, nonnative structures, e.g. aggregates, can form rapidly at low denaturant concentrations, and their subsequent conversion to the native structure is slow.     

A comparison of the folding of two knotted proteins: YbeA and YibK

The extraordinary topology of proteins belonging to the alpha/beta-knot superfamily of proteins is unexpected, due to the apparent complexities involved in the formation of a deep trefoil knot in a polypeptide backbone. Despite this, an increasing number of knotted structures are being identified; how such proteins fold remains a mystery. Studies on the dimeric protein YibK from Haemophilus influenzae have led to the characterisation of its folding pathway in some detail. To complement research into the folding of YibK, and to address whether folding pathways are conserved for members of the alpha/beta-knot superfamily, the structurally similar knotted protein YbeA from Escherichia coli has been studied. A comprehensive thermodynamic and kinetic analysis of the folding of YbeA is presented here, and compared to that of YibK. Both fold via an intermediate state populated under equilibrium conditions that is monomeric and considerably structured. The unfolding/refolding kinetics of YbeA are simpler than those found for YibK and involve two phases attributed to the formation of a monomeric intermediate state and a dimerisation step. In contrast to YibK, a change in the rate-determining step on the unfolding pathway for YbeA is observed with a changing concentration of urea. Despite this difference, both proteins fold by a mechanism involving at least one sequential monomeric intermediate that has properties similar to that observed during the equilibrium unfolding. The rate of dimerisation observed for YbeA and YibK is very similar, as is the rate constant for formation of the kinetic monomeric intermediate that precedes dimerisation. The findings suggest that relatively slow folding and dimerisation may be common attributes of knotted proteins.     

The effect of Spo0A and AbrB proteins on expression of the genes of guanyl-specific ribonucleases from Bacillus intermedius and Bacillus pumilus in Bacillus subtilis recombinant strains

Guanyl-specific ribonucleases from Bacillus intermedius and Bacillus pumilus are actively secreted under phosphate starvation by recombinant strains of Bacillus subtilis with native regulatory systems and by strains defective in some proteins of the Spo0A phosphorylation pathway. The level of expression of ribonuclease genes has been shown to increase approximately sixfold in recombinant strains with mutation in the spo0A gene and threefold in the spo0A/abrB mutants, as compared with native strains. These results demonstrate that the Spo0A protein regulates the production of ribonucleases and thus acts as a repressor, while the AbrB protein is an activator of expression of the genes encoding ribonucleases from Bacillus intermedius and Bacillus pumilus in Bacillus subtilis cells.     

Non-chromatographic strategies for protein refolding

Overexpression of recombinant proteins in bacterial systems (such as E. coli) often leads to formation of inactive and insoluble ' inclusion bodies' . Protein refolding refers to folding back the proteins after solubilizing/unfolding the misfolded proteins of the inclusion bodies. Protein aggregation, a concentration dependent phenomenon, competes with refolding pathway. The refolding strategies largely aim at reducing aggregation and/or promoting correct folding. This review focuses on non-chromatographic strategies for refolding like dilution, precipitation, three phase partitioning and macro-(affinity ligand) facilitated three phase partitioning. The nanomaterials which disperse well in aqueous buffers are also discussed in the context of facilitating protein refolding. Apart from general results with these methods, the review also covers the use of non-chromatographic methods in protein refolding in the patented literature beyond 2000. The patented literature generally describes use of cocktail of additives which results in increase in refolding yield. Such additives include low concentration of chaotropic agents, redox systems, ions like SO4(2-) and Cl-, amines, carboxylic acids and surfactants. Some novel approaches like use of a "pressure window" or ionic liquids for refolding and immobilized diselenide compounds for ensuring correct -S-S- bonds pairing have also been discussed in various patents. In most of the patented literature, focus naturally has been on refolding in case of pharmaceutical proteins.     

Characterization of the folding and unfolding reactions of single-chain monellin: evidence for multiple intermediates and competing pathways

The mechanisms of folding and unfolding of the small plant protein monellin have been delineated in detail. For this study, a single-chain variant of the natively two-chain monellin, MNEI, was used, in which the C terminus of chain B was connected to the N terminus of chain A by a Gly-Phe linker. Equilibrium guanidine hydrochloride (GdnHCl)-induced unfolding experiments failed to detect any partially folded intermediate that is stable enough to be populated at equilibrium to a significant extent. Kinetic experiments in which the refolding of GdnHCl-unfolded protein was monitored by measurement of the change in the intrinsic tryptophan fluorescence of the protein indicated the accumulation of three transient partially structured folding intermediates. The fluorescence change occurred in three kinetic phases: very fast, fast, and slow. It appears that the fast and slow changes in fluorescence occur on competing folding pathways originating from one unfolded form and that the very fast change in fluorescence occurs on a third parallel pathway originating from a second unfolded form of the protein. Kinetic experiments in which the refolding of alkali-unfolded protein was monitored by the change in the fluorescence of the hydrophobic dye 8-anilino-1-naphthalenesulfonic acid (ANS), consequent to the dye binding to the refolding protein, as well as by the change in intrinsic tryptophan fluorescence, not only confirmed the presence of the three kinetic intermediates but also indicated the accumulation of one or more early intermediates at a few milliseconds of refolding. These experiments also exposed a very slow kinetic phase of refolding, which was silent to any change in the intrinsic tryptophan fluorescence of the protein. Hence, the spectroscopic studies indicated that refolding of single-chain monellin occurs in five distinct kinetic phases. Double-jump, interrupted-folding experiments, in which the accumulation of folding intermediates and native protein during the folding process could be determined quantitatively by an unfolding assay, indicated that the fast phase of fluorescence change corresponds to the accumulation of two intermediates of differing stabilities on competing folding pathways. They also indicated that the very slow kinetic phase of refolding, identified by ANS binding, corresponds to the formation of native protein. Kinetic experiments in which the unfolding of native protein in GdnHCl was monitored by the change in intrinsic tryptophan fluorescence indicated that this change occurs in two kinetic phases. Double-jump, interrupted-unfolding experiments, in which the accumulation of unfolding intermediates and native protein during the unfolding process could be determined quantitatively by a refolding assay, indicated that the fast unfolding phase corresponds to the formation of fully unfolded protein via one unfolding pathway and that the slow unfolding phase corresponds to a separate unfolding pathway populated by partially unfolded intermediates. It is shown that the unfolded form produced by the fast unfolding pathway is the one which gives rise to the very fast folding pathway and that the unfolded form produced by the slower unfolding pathway is the one which gives rise to the slow and fast folding pathways.     

Partially oxidized active intermediates in refolding of reduced ribonuclease

The refolding of reduced ribonuclease A has been studied by measurements of enzymatic activity under conditions where the oxidation of thiol groups into disulfide bonds is rather slow. The sensitivity to a treatment by N-ethylmaleimide has been used to distinguish between partially and totally oxidized active species. It is found that the first active protein molecules to be formed do not have all of their disulfide bonds. Because they are active, these partially oxidized intermediates probably have very close to native conformation, which they can reach without being trapped in a wrong structure by forming too many incorrect disulfide bonds. The significance of these intermediates to the refolding pathway of reduced ribonuclease is discussed.     

Aggregation events occur prior to stable intermediate formation during refolding of interleukin 1beta

A point mutation, lysine 97 --> isoleucine (K97I), in a surface loop in the beta-sheet protein interleukin 1beta (IL-1beta), exhibits increased levels of inclusion body (IB) formation relative to the wild-type protein (WT) when expressed in Escherichia coli. Despite the common observation that less stable proteins are often found in IBs, K97I is more stable than WT. We examined the folding pathway of the mutant and wild-type proteins at pH 6.5 and 25 degrees C with manual-mixing and stopped-flow optical spectroscopy to determine whether changes in the properties of transiently populated species in vitro correlate with the observation of increased aggregation in vivo. The refolding reactions of the WT and K97I proteins are both described by three exponential processes. Two exponential processes characterize fast events (0.1-1.0 s) in folding while the third exponential process correlates with a slow (70 s) single pathway to and from the native state. The K97I replacement affects the earlier steps in the refolding pathway. Aggregation, absent in the WT refolding reaction, occurs in K97I above a critical protein concentration of 18 microM. This observation is consistent with an initial nucleation step mediating protein aggregation. Stopped-flow kinetic studies of the K97I aggregation process demonstrate that K97I aggregates most rapidly during the earliest refolding times, when unfolded protein conformers remain highly populated and the concentration of folding intermediates is low. Folding and aggregation studies together support a model in which the formation of stable folding intermediates afford protection against further K97I aggregation.     

Protein aggregation as bacterial inclusion bodies is reversible

Inclusion bodies are refractile, intracellular protein aggregates usually observed in bacteria upon targeted gene overexpression. Since their occurrence has a major economical impact in protein production bio-processes, in vitro refolding strategies are under continuous exploration. In this work, we prove spontaneous in vivo release of both beta-galactosidase and P22 tailspike polypeptides from inclusion bodies resulting in their almost complete disintegration and in the concomitant appearance of soluble, properly folded native proteins with full biological activity. Since, in particular, the tailspike protein exhibits an unusually slow and complex folding pathway involving deep interdigitation of beta-sheet structures, its in vivo refolding indicates that bacterial inclusion body proteins are not collapsed into an irreversible unfolded state. Then, inclusion bodies can be observed as transient deposits of folding-prone polypeptides, resulting from an unbalanced equilibrium between in vivo protein precipitation and refolding that can be actively displaced by arresting protein synthesis. The observation that the formation of big inclusion bodies is reversible in vivo can be also relevant in the context of amyloid diseases, in which deposition of important amounts of aggregated protein initiates the pathogenic process.     

蛋白质的氧化重折叠

经过近几十年来广泛而深入的研究,蛋白质氧化重折叠的机制已得到相当详细的阐明。1在已研究过的蛋白质中,大多数蛋白质都是沿着多途径而非单一、特定的途径进行氧化重折叠,这与折叠能量景观学说是一致的。2正是氨基酸残基间的天然相互作用而不是非天然的相互作用控制蛋白质的折叠过程。这一结论与含非天然二硫键的折叠中间体在牛胰蛋白酶抑制剂（BPTI）折叠中所起的重要作用并非相互排斥,因为后者仅仅是进行链内二硫键重排的化学反应所必需,与控制肽链折叠无直接关系。3根据对BPTI的研究,二硫键曾被认为仅仅具有稳定蛋白质天然结构的作用,既不决定折叠途径也不决定其三维构象。这一观点不适用于其它蛋白质。对凝乳酶原的研究表明,天然二硫键的形成是恢复天然构象的前提。天然二硫键的形成与肽键的正确折叠相辅相成,更具有普遍意义。4在氧化重折叠的早期,二硫键的形成基本上是一个随机过程,随着肽链的折叠二硫键的形成越来越受折叠中间体构象的限制。提高重组蛋白质的复性产率是生物技术领域中的一个巨大的挑战。除了分子聚集外,在折叠过程中所形成的二硫键错配分子是导致低复性率的另一个主要原因。氧化重折叠机制的阐明为解决此问题提供了有益的启示。如上所述,在折叠的后期,二硫键的形成决定于折叠中间体的构象,类天然、有柔性的结构有利于天然二硫键形成和正确折叠,具有这类结构的分子为有效的折叠中间体,最终都能转变为天然产物;而无效折叠中间体往往具有稳定的结构,使巯基、二硫键内埋妨碍二硫键重排,并因能垒的障碍不利于进一步折叠。因此,降低无效折叠中间体的稳定性使之转变为有效折叠中间体是提高含二硫键蛋白质复性率的一条基本原则,实验证明,碱性pH、低温、降低蛋白质稳定性的试剂、蛋白质二硫键异构酶、改变蛋白质一级结构是实现这一原则的有效手段。此外,这里还就氧化重折叠的基础和应用研究的前景进行了讨论。