Functional Refolding Research Articles

Functional refolding of the penetration protein on a non-enveloped virus.

Non-enveloped viruses must create a transient membrane lesion to initiate infection by transferring their genomes into a target cell1. Rotaviruses offer a particularly favorable opportunity to visualize the mechanism for subviral particle delivery, the principal function of their outer-layer protein, VP42–4. We show here by electron cryomicroscopy (cryo-EM) that VP4, activated by cleavage to VP8* and VP5*, rearranges on the virion surface from an “upright” to a “reversed” conformation. The reversed structure projects an initially buried, “foot” domain outward into the host cell membrane to which the virion has attached. Analysis of cryo-tomograms of virus particles entering cells is consistent with this picture. We have stabilized with a disulfide mutant a likely intermediate in this transition. The results define molecular mechanisms for the first steps in viral membrane penetration and suggest similarities with mechanisms postulated for other viruses.

The Conformational Equilibrium of the Neuropeptide Y2 Receptor in Bilayer Membranes.

Dynamic structural transitions within the seven‐transmembrane bundle represent the mechanism by which G‐protein‐coupled receptors convert an extracellular chemical signal into an intracellular biological function. Here, the conformational dynamics of the neuropeptide Y receptor type 2 (Y2R) during activation was investigated. The apo, full agonist‐, and arrestin‐bound states of Y2R were prepared by cell‐free expression, functional refolding, and reconstitution into lipid membranes. To study conformational transitions between these states, all six tryptophans of Y2R were 13C‐labeled. NMR‐signal assignment was achieved by dynamic‐nuclear‐polarization enhancement and the individual functional states of the receptor were characterized by monitoring 13C NMR chemical shifts. Activation of Y2R is mediated by molecular switches involving the toggle switch residue Trp2816.48 of the highly conserved SWLP motif and Trp3277.55 adjacent to the NPxxY motif. Furthermore, a conformationally preserved “cysteine lock”‐Trp11623.50 was identified.

Improved in Vitro Folding of the Y2 G Protein-Coupled Receptor into Bicelles.

Prerequisite for structural studies on G protein-coupled receptors is the preparation of highly concentrated, stable, and biologically active receptor samples in milligram amounts of protein. Here, we present an improved protocol for Escherichia coli expression, functional refolding, and reconstitution into bicelles of the human neuropeptide Y receptor type 2 (Y2R) for solution and solid-state NMR experiments. The isotopically labeled receptor is expressed in inclusion bodies and purified using SDS. We studied the details of an improved preparation protocol including the in vitro folding of the receptor, e.g., the native disulfide bridge formation, the exchange of the denaturating detergent SDS, and the functional reconstitution into bicelle environments of varying size. Full pharmacological functionality of the Y2R preparation was shown by a ligand affinity of 4 nM and G-protein activation. Further, simple NMR experiments are used to test sample quality in high micromolar concentration.

Modulating bilayer mechanical properties to promote the coupled folding and insertion of an integral membrane protein.

Bilayer mechanical properties are not only of crucial importance to the mechanism of action of mechanosensation in lipid membranes but also affect preparative laboratory taskssuch as membrane-protein refolding. We report this for coupled refolding and bilayer insertion of outer membrane phospholipaseA (OmpLA), an integral membrane enzyme that catalyses the hydrolytic cleavage of glycerophospholipids. OmpLA can be refolded into a variety of detergent micelles and unilamellar vesicles composed of short-chain phospholipids but, in the absence of chemical or molecular chaperones, not into thicker membranes. Controlled modulation of bilayer mechanical properties by judicious use of subsolubilising concentrations of detergents induces monolayer curvature strain, acyl chain fluidisation, membrane thinning, and transient aqueous bilayer defects. This enables quantitative and functional refolding of OmpLA even into bilayer membranes composed of long-chain phospholipids to yield enzymatically active proteoliposomes without requiring membrane solubilisation.

A fluorinated detergent for membrane-protein applications.

Surfactants carrying fluorocarbon chains hold great promise as gentle alternatives to conventional hydrocarbon-based detergents for the solubilization and handling of integral membrane proteins. However, their inertness towards lipid bilayer membranes has limited the usefulness of fluorinated surfactants in situations where detergent-like activity is required. We demonstrate that fluorination does not necessarily preclude detergency, as exemplified by a fluorinated octyl maltoside derivative termed F6 OM. This nonionic compound readily interacts with and completely solubilizes phospholipid vesicles in a manner reminiscent of conventional detergents without, however, compromising membrane order at subsolubilizing concentrations. Owing to this mild and unusual mode of detergency, F6 OM outperforms a lipophobic fluorinated surfactant in chaperoning the functional refolding of an integral membrane enzyme by promoting bilayer insertion in the absence of micelles.

Refolding and Enzyme Kinetic Studies on the Ferrochelatase of the Cyanobacterium Synechocystis sp. PCC 6803

Heme is a cofactor for proteins participating in many important cellular processes, including respiration, oxygen metabolism and oxygen binding. The key enzyme in the heme biosynthesis pathway is ferrochelatase (protohaem ferrolyase, EC 4.99.1.1), which catalyzes the insertion of ferrous iron into protoporphyrin IX. In higher plants, the ferrochelatase enzyme is localized not only in mitochondria, but also in chloroplasts. The plastidic type II ferrochelatase contains a C-terminal chlorophyll a/b (CAB) motif, a conserved hydrophobic stretch homologous to the CAB domain of plant light harvesting proteins and light-harvesting like proteins. This type II ferrochelatase, found in all photosynthetic organisms, is presumed to have evolved from the cyanobacterial ferrochelatase. Here we describe a detailed enzymological study on recombinant, refolded and functionally active type II ferrochelatase (FeCh) from the cyanobacterium Synechocystis sp. PCC 6803. A protocol was developed for the functional refolding and purification of the recombinant enzyme from inclusion bodies, without truncation products or soluble aggregates. The refolded FeCh is active in its monomeric form, however, addition of an N-terminal His6-tag has significant effects on its enzyme kinetics. Strikingly, removal of the C-terminal CAB-domain led to a greatly increased turnover number, kcat, compared to the full length protein. While pigments isolated from photosynthetic membranes decrease the activity of FeCh, direct pigment binding to the CAB domain of FeCh was not evident.

Functional Refolding and Characterization of Two Tom40 Isoforms from Human Mitochondria

Tom40 proteins represent an essential class of molecules which facilitate translocation of unfolded proteins from the cytosol into the mitochondrial intermembrane space. They are part of a high-molecular mass complex that forms the protein-conducting channel in outer mitochondrial membranes. This study concerns the recombinant expression, purification and folding of amino-terminally truncated variants of the two human Tom40 isoforms for structural biology experiments. Both CD and FTIR secondary structure analysis revealed a dominant beta-sheet structure and a short alpha-helical part for both proteins together with a high thermal stability. Two secondary structure elements can be denatured independently. Reconstitution of the recombinant protein into planar lipid bilayers demonstrated ion channel activity similar to Tom40 purified from Neurospora crassa mitochondrial membranes, but conductivity fingerprints differ from the structurally closely related VDAC proteins.

Mapping of Disulfide Bonds within the Amino-terminal Extracellular Domain of the Inhibitory Glycine Receptor

The strychnine-sensitive glycine receptor (GlyR) is a ligand-gated chloride channel and a member of the superfamily of cysteine loop (Cys-loop) neurotransmitter receptors, which also comprises the nicotinic acetylcholine receptor (nAChR). Within the extracellular domain (ECD), the eponymous Cys-loop harbors two conserved cysteines, assumed to be linked by a superfamily-specific disulfide bond. The GlyR ECD carries three additional cysteine residues, two are predicted to form a second, GlyR-specific bond. The configuration of none of the cysteines of GlyR, however, had been determined directly. Based on a crystal structure of the nAChRalpha1 ECD, we generated a model of the human GlyRalpha1 where close proximity of the respective cysteines was consistent with the formation of both the Cys-loop and the GlyR-specific disulfide bonds. To identify native disulfide bonds, the GlyRalpha1 ECD was heterologously expressed and refolded under oxidative conditions. By matrix-assisted laser desorption ionization time-of-flight mass spectrometry, we detected tryptic fragments of the ECD indicative of disulfide bond formation for both pairs of cysteines, as proposed by modeling. The identity of tryptic fragments was confirmed using chemical modification of cysteine and lysine residues. As evident from circular dichroism spectroscopy, mutagenesis of single cysteines did not impair refolding of the ECD in vitro, whereas it led to partial or complete intracellular retention and consequently to a loss of function of full-length GlyR subunits in human embryonic kidney 293 cells. Our results indicate that the GlyR ECD forms both a Cys-loop and a GlyR-specific disulfide bond. In addition, cysteine residues appear to be important for protein maturation in vivo.

Allostery and functional refolding in the Gram-negative hexameric Type II citrate synthases

Allostery and functional refolding in the Gram-negative hexameric Type II citrate synthases

Effects of divalent cations on encapsulation and release in the GroEL-assisted folding

Chaperonin GroEL assists protein folding in the presence of ATP and magnesium. Recent studies have shown that several divalent cations other than magnesium induce conformational changes of GroEL, thereby influencing chaperonin-assisted protein folding, but little is known about the detailed mechanism for such actions. Thus, the effects of divalent cations on protein encapsulation by GroEL/ES complexes were investigated. Of the divalent cations, not only magnesium, but also manganese ions enabled the functional refolding and release of 5,10-methylenetetrahydroforate reductase (METF) by GroEL. Neither ATP hydrolysis nor METF refolding was observed in the presence of zinc ion, whereas only ATP hydrolysis was induced by cobalt and nickel ions. SDS-PAGE and gel filtration analyses revealed that cobalt, nickel and zinc ions permit the formation of stable substrate-GroEL-GroES cis-ternary complexes, but prevent the release of METF from GroEL.

Functional refolding of a recombinant C-type lectin-like domain containing intramolecular disulfide bonds

The lectin-like oxidized low-density lipoprotein scavenger receptor (LOX-1) is a pro-inflammatory marker and Type II membrane protein expressed on vascular cells and tissues. The LOX-1 extracellular domain mediates recognition of oxidized low-density lipoprotein (oxLDL) particles that are implicated in the development of atherosclerotic plaques. To study the molecular basis for LOX-1-mediated ligand recognition, we have expressed, purified and refolded a recombinant LOX-1 protein and assayed for its biological activity using a novel fluorescence-based assay to monitor binding to lipid particles. Overexpression of a hexahistidine-tagged cysteine-rich LOX-1 extracellular domain in bacteria leads to the formation of aggregates that accumulated in bacterial inclusion bodies. The hexahistidine-tagged LOX-1 molecule was purified by affinity chromatography from solubilized inclusion bodies. A sequential dialysis procedure was used to refold the purified but inactive and denatured LOX-1 protein into a functionally active form that mediated recognition of oxLDL particles. This approach allowed slow LOX-1 refolding and assembly of correct intrachain disulfide bonds. Circular dichroism analysis of the refolded LOX-1 molecule demonstrated a folded state with substantial α-helical content. Using immobilized recombinant, refolded LOX-1 we demonstrated a 70-fold preferential recognition for oxLDL over native LDL particles. Thus, a protein domain containing intrachain disulfide bonds can be reconstituted into a functionally active state using a relatively simple dialysis-based technique.

Functional refolding of the Campylobacter jejuni MOMP (major outer membrane protein) porin by GroEL from the same species.

Functional and structural studies of outer membrane proteins from Gram-negative bacteria are frequently carried out using refolded proteins. Recombinant proteins are produced in Escherichia coli as inclusion bodies and then tediously refolded by dilution in buffered detergent solutions. In the present work, we obtained the refolding of MOMP (major outer membrane protein) from Campylobacter assisted by the molecular chaperone GroEL. Refolded MOMP recovered its native pore-forming activity when reconstituted in planar lipid bilayers. Both proteins were purified from the Campylobacter jejuni strain 85H. The purity of GroEL was assessed by silver staining and MS. Its native ultrastructure was observed by the use of transmission electron microscopy. Denaturation of MOMP was performed in urea at 65 degrees C followed by dialysis against 100 mM acetic acid, and was assessed by CD analysis. MOMP refolding reached a maximum efficiency in the presence of GroEL (at a MOMP/GroEL molar ratio of 9:1) and ATP. Under these conditions, 95% of denatured MOMP was refolded after a 15 min incubation. This approach represents an alternative method in studies of membrane protein refolding.

Leishmania donovani methionine adenosyltransferase. Role of cysteine residues in the recombinant enzyme.

Methionine adenosyltransferase (MAT, EC 2.5.1.6)-mediated synthesis of S-adenosylmethionine (AdoMet) is a two-step process consisting of the formation of AdoMet and the subsequent cleavage of the tripolyphosphate (PPPi) molecule, a reaction induced, in turn, by AdoMet. The fact that the two activities, AdoMet synthesis and tripolyphosphate hydrolysis, can be measured separately is particularly useful when the site-directed mutagenesis approach is used to determine the functional role of the amino acid residues involved in each. The present report describes the cloning and subsequent functional refolding, using a bacterial expression system, of the MAT gene (GenBank accession number AF179714) from Leishmania donovani, the etiological agent of visceral leishmaniasis. The absolute need to include a sulfhydryl-protection reagent in the refolding buffer for this protein, in conjunction with the rapid inactivation of the functionally refolded protein by N-ethylmaleimide, suggests the presence of crucial cysteine residues in the primary structure of the MAT protein. The seven cysteines in L. donovani MAT were mutated to their isosterical amino acid, serine. The C22S, C44S, C92S and C305S mutants showed a drastic loss of AdoMet synthesis activity compared to the wild type, and the C33S and C47S mutants retained a mere 12% of wild-type MAT activity. C106S mutant activity and kinetics remained unchanged with respect to the wild-type. Cysteine substitutions also modified PPPi cleavage and AdoMet induction. The C22S, C44S and C305S mutants lacked in tripolyphosphatase activity altogether, whereas C33S, C47S and C92S retained low but detectable activity. The behavior of the C92S mutant was notable: its inability to synthesize AdoMet combined with its retention of tripolyphosphatase activity appear to be indicative of the specific involvement of the respective residue in the first step of the MAT reaction.

Interaction of the Eukaryotic Elongation Factor 1A with Newly Synthesized Polypeptides

eEF1A, the eukaryotic homologue of bacterial elongation factor Tu, is a well characterized translation elongation factor responsible for delivering aminoacyl-tRNAs to the A-site at the ribosome. Here we show for the first time that eEF1A also associates with the nascent chain distal to the peptidyltransferase center. This is demonstrated for a variety of nascent chains of different lengths and sequences. Interestingly, unlike other ribosome-associated factors, eEF1A also interacts with polypeptides after their release from the ribosome. We demonstrate that eEF1A does not bind to correctly folded full-length proteins but interacts specifically with proteins that are unable to fold correctly in a cytosolic environment. This association was demonstrated both by photo-cross-linking and by a functional refolding assay.

Refolding of recombinant alpha and beta subunits of the Rhodospirillum rubrum F(0)F(1) ATP synthase into functional monomers that reconstitute an active alpha(1)beta(1)-dimer.

The alpha subunit from the Rhodospirillum rubrum F(0)F(1) ATP synthase (RrF(1)alpha) was over-expressed in unc operon-deleted Escherichia coli strains under various growth conditions only in insoluble inclusion bodies. The functional refolding of urea-solubilized RrF(1)alpha was followed by measuring its ability to stimulate the restoration of ATP synthesis and hydrolysis in beta-less R. rubrum chromatophores reconstituted with pure native or recombinant RrF(1)beta [Nathanson, L. & Gromet-Elhanan, Z. (1998) J. Biol. Chem. 273, 10933-10938]. The refolding efficiency was found to increase with decreasing RrF(1)alpha concentrations and required high concentrations of MgATP, saturating approximately 60% when 50 microgram protein.mL(-1) were refolded in presence of 50 mM MgATP. Size-exclusion HPLC of such refolded RrF(1)alpha revealed a 50-60% decrease in its aggregated form and a parallel appearance of its monomeric peak. RrF(1)beta refolded under identical conditions appeared almost exclusively as a monomer. This procedure enabled the isolation of large amounts of a stable RrF(1)alpha monomer, which stimulated the restoration of ATP synthesis and hydrolysis much more efficiently than the refolded alpha mixture, and bound ATP and ADP in a Mg-dependent manner. Incubation of both RrF(1)alpha and beta monomers, which by themselves had no ATPase activity, resulted in a parallel appearance of activity and assembled alpha(1)beta(1)-dimers, but showed no formation of alpha(3)beta(3)-hexamers. The RrF(1)-alpha(1)beta(1)-ATPase activity was, however, very similar to the activity observed in isolated native chloroplast CF(1)-alpha(3)beta(3), indicating that these dimers contain only the catalytic nucleotide-binding site at their alpha/beta interface. Their inability to associate into an alpha(3)beta(3)-hexamer seems therefore to reflect a much lower stability of the noncatalytic RrF(1) alpha/beta interface.

Molecular chaperones protect catalase against thermal stress.

Lenticular alpha-crystallin is generally thought of as having limited chaperone functions. It can efficiently suppress the aggregation of proteins but is unable to promote the functional refolding of proteins after denaturation in many systems unlike other molecular chaperones. However, it has been reported that alpha-crystallin, along with the small heat-shock proteins, is able to promote the functional refolding of some enzymes after thermal and chemical denaturation. These chaperones are also able to confer protection against the thermal inactivation of these enzymes. In results presented here, we demonstrate that alpha-crystallin, along with chaperonin 60 (GroEL), was able to provide statistically significant and specific protection against catalase thermal inactivation at stoichiometrical concentrations. The small heat-shock protein, heat-shock protein 25 (Hsp25), was unable to confer any such protection. alpha-Crystallin however was unable to promote the functional refolding of thermally inactivated catalase. alpha-Crystallin and Hsp25 both efficiently suppressed the thermal aggregation of catalase. A high-molecular-mass (HMM) complex was only observed to develop in solutions containing catalase and alpha-crystallin after solutions were 80-fold more concentrated relative to thermal inactivation assay conditions prior to incubation. SDS/PAGE analysis confirmed that alpha-crystallin had formed a soluble complex with catalase after a period of thermal stress.

Method for increasing the yield of properly folded recombinant human gamma interferon from inclusion bodies

A strategy is described for improved refolding and purification of recombinant human gamma interferon (rh-IFN γ), which could warrant a higher yield and specific activity than reported previously. The optimal conditions of refolding are obtained by addition of a labilizing agent, l-arginine, in the refolding buffer. A 10-fold increase in the yield was observed with 0.5 M l-arginine, compared with renaturation in its absence. By varying renaturation parameters, the conditions that allow functional refolding of ∼25–30% of the recombinant protein have been standardized. A simple process is also described for the purification of rh-IFN γ. The purification involves a single-column chromatography on S-Sepharose, after refolding of rh-IFN γ in arginine containing buffer. This procedure has consistently produced rh-IFN γ having a purity of at least 97%, the rest being the aggregated form of gamma interferon. The purified protein is a dimer under non-denaturing conditions and has a specific activity of 2×10 8 IU mg −1 protein, as measured by viral cytopathic assay.

Cell-free synthesis, functional refolding, and spectroscopic characterization of bacteriorhodopsin, an integral membrane protein.

Bacteriorhodopsin (bR) is an integral membrane protein which functions as a light-driven proton pump in Halobacterium halobium (also known as Halobacterium salinarium). The cell-free synthesis of bR in quantities sufficient for FTIR and NMR spectroscopy and the ability to selectively isotope label bR using aminoacylated suppressor tRNAs would provide a powerful approach for studying the role of specific amino acid residues. However, no integral membrane protein has yet been expressed in a cell-free system in quantities sufficient for such biophysical studies. We report the cell-free synthesis of bacterioopsin, its purification, its refolding in polar lipids from H. halobium, and its regeneration with all-trans-retinal to yield bacteriorhodopsin in a form functionally similar to bR in purple membrane. Importantly, the yields obtained from in vitro and in vivo expression are comparable. Functionality of the cell-free expressed bR is established using static and time-resolved absorption spectroscopy and FTIR difference spectroscopy.

Small heat shock proteins are molecular chaperones.

Small heat shock proteins (sHsp) with a molecular mass of 15-30 kDa are ubiquitous and conserved. Up to now their function has remained enigmatic. Increased expression under heat shock conditions and their protective effect on cell viability at elevated temperatures suggest that they may have a function in the formation or maintenance of the native conformation of cytosolic proteins. To test this hypothesis we studied the influence of murine Hsp25, human Hsp27, and bovine alpha-B-crystallin (an eye lens protein homologous to sHsps) on the unfolding and refolding of citrate synthase and alpha-glucosidase in vitro. Here we show that all sHsps investigated act as molecular chaperones in these folding reactions. At stoichiometric amounts they maximally prevent the aggregation of citrate synthase and alpha-glucosidase under heat shock conditions and stabilize the proteins. Furthermore, they promote the functional refolding of these proteins after urea denaturation similar to GroE and Hsp90. The interaction both with unfolding and refolding proteins seems to be ATP-independent.

Interaction of GroE with an all-beta-protein.

Molecular chaperones are involved in protein folding both in vivo and in vitro. The Escherichia coli chaperone GroEL interacts with a number of nonnative proteins. A common structural motif of nonnative proteins, which is recognized by GroEL, has not yet been identified. In order to study the role of beta-sheet secondary structure on the interaction of nonnative proteins with GroEL, we used the F(ab) fragment of a monoclonal antibody as a model substrate protein. Here we show that GroEL interacts functionally with this all-beta-protein during reactivation. Antibody fragments refold spontaneously in good yield from the guanidine-denatured state. Functional refolding to the native state is inhibited transiently by GroEL, but there is no complete folding arrest in the absence of Mg-ATP and GroES. The yield of these unspecifically released GroEL-bound F(ab) fragments corresponds to that of the spontaneous reactivation in the absence of chaperones. However, the refolding kinetics in the presence of GroEL are considerably slower. The addition of Mg-ATP to the GroEL.F(ab) complex results in an immediate release of bound substrate protein and a significant increase in the amount of reconstituted antibody fragments compared to spontaneous reactivation. GroES is not essential for functional GroEL-mediated refolding of the F(ab) fragment but affects the reactivation yield to a small extent. Interestingly, stimulation of the GroEL-mediated F(ab) refolding depends primarily on the binding and not on hydrolysis of adenosine triphosphates. Previous results indicate the binding of alpha-helices to GroEL. The results presented in this paper suggest that beta-sheet secondary structural elements are recognized by GroEL. We therefore conclude that the interaction of a nonnative protein with GroEL depends mainly on the nature of the early folding intermediate but not on a specific element of secondary structure.