Refolding Reaction Research Articles

4-Thiaproline accelerates the slow folding phase of proteins containing cis prolines in the native state by two orders of magnitude.

The cis/trans isomerization of peptidyl-prolyl peptide bonds is often the bottleneck of the refolding reaction for proteins containing cis proline residues in the native state. Proline (Pro) analogues, especially C4-substituted fluoroprolines, have been widely used in protein engineering to enhance the thermodynamic stability of peptides and proteins and to investigate folding kinetics. 4-thiaproline (Thp) has been shown to bias the ring pucker of Pro, to increase the cis population percentage of model peptides in comparison to Pro, and to diminish the activation energy barrier for the cis/trans isomerization reaction. Despite its intriguing properties, Thp has been seldom incorporated into proteins. Moreover, the impact of Thp on the folding kinetics of globular proteins has never been reported. In this study, we show that upon incorporation of Thp at cisPro76 into the thioredoxin variant Trx1P the half-life of the refolding reaction decreased from ~2 h to ~35 s. A dramatic acceleration of the refolding rate could be observed also for the protein pseudo wild-type barstar upon replacement of cisPro48 with Thp. Quantum chemical calculations suggested that the replacement of the Cγ H2 group by a sulfur atom in the pyrrolidine ring, might lower the barrier for cis/trans rotation due to a weakened peptide bond. The protein variants retained their thermodynamic stability upon incorporation of Thp, while the catalytic and enzymatic activities of the modified Trx1P remained unchanged. Our results show that the Pro isostere Thp might accelerate the rate of the slow refolding reaction for proteins containing cis proline residues in the native state, independent from the local structural environment.

An all-in-one state-observer for protein refolding reactions using particle filters and delayed measurements

Proper monitoring as basis for process optimization and control of protein refolding reactions in real-time is difficult and currently available techniques are either expensive, not applicable in real-time or give only limited information about the ongoing process. Model-based methods such as particle filters (PFs) have been used in different biological systems for state-estimation to overcome difficulties arising from states that are hard or impossible to measure, often low measurement frequencies and high measurement delay. Since recent approaches had difficulties to overcome all these problems, a novel approach via a PF including a mechanistic model is used. The PF is calibrated and tuned with experimental data and its applicability validated with two additional experiments. It is shown how augmentation of model parameters can be used for state-estimation in real-time to better adapt to model inaccuracies, poor model calibration or application of the calibrated model to a new process. Furthermore, it is shown that the PF can deal with low measurement frequencies and high measurement delay, resulting in reliable tracking of the process with normalized root mean squared errors (NRMSE) of the native protein and folding intermediates between 3.44 and 6.62%, values in the range of 18 to 93% less compared to simple feed-forward simulation.

MediMer: a versatile do-it-yourself peptide-receptive MHC class I multimer platform for tumor neoantigen-specific T cell detection.

Peptide-loaded MHC class I (pMHC-I) multimers have revolutionized our capabilities to monitor disease-associated T cell responses with high sensitivity and specificity. To improve the discovery of T cell receptors (TCR) targeting neoantigens of individual tumor patients with recombinant MHC molecules, we developed a peptide-loadable MHC class I platform termed MediMer. MediMers are based on soluble disulfide-stabilized β2-microglobulin/heavy chain ectodomain single-chain dimers (dsSCD) that can be easily produced in large quantities in eukaryotic cells and tailored to individual patients' HLA allotypes with only little hands-on time. Upon transient expression in CHO-S cells together with ER-targeted BirA biotin ligase, biotinylated dsSCD are purified from the cell supernatant and are ready to use. We show that CHO-produced dsSCD are free of endogenous peptide ligands. Empty dsSCD from more than 30 different HLA-A,B,C allotypes, that were produced and validated so far, can be loaded with synthetic peptides matching the known binding criteria of the respective allotypes, and stored at low temperature without loss of binding activity. We demonstrate the usability of peptide-loaded dsSCD multimers for the detection of human antigen-specific T cells with comparable sensitivities as multimers generated with peptide-tethered β2m-HLA heavy chain single-chain trimers (SCT) and wild-type peptide-MHC-I complexes prior formed in small-scale refolding reactions. Using allotype-specific, fluorophore-labeled competitor peptides, we present a novel dsSCD-based peptide binding assay capable of interrogating large libraries of in silico predicted neoepitope peptides by flow cytometry in a high-throughput and rapid format. We discovered rare T cell populations with specificity for tumor neoepitopes and epitopes from shared tumor-associated antigens in peripheral blood of a melanoma patient including a so far unreported HLA-C*08:02-restricted NY-ESO-1-specific CD8+ T cell population. Two representative TCR of this T cell population, which could be of potential value for a broader spectrum of patients, were identified by dsSCD-guided single-cell sequencing and were validated by cognate pMHC-I multimer staining and functional responses to autologous peptide-pulsed antigen presenting cells. By deploying the technically accessible dsSCD MHC-I MediMer platform, we hope to significantly improve success rates for the discovery of personalized neoepitope-specific TCR in the future by being able to also cover rare HLA allotypes.

Quantitative analytics for protein refolding states

Development and optimization of inclusion body (IB) refolding processes is often based on empirical strategies. Thus, generation of in-depth process understanding is crucial to shift towards more knowledge-driven approaches. Within this contribution the goal was to establish a systematic approach for the reliable quantification of process dynamics in protein refolding processes. Established offline- analytical tools like HPLC-based methods or photometric assays were analyzed regarding their specifications and consequent suitability to quantify protein states. An in-silico study was conducted to define requirements for state quantification by analyzing the influences of error, discrete sampling intervals and the pace of the reaction on signal quality. Refolding and aggregation reaction rates were calculated by finite difference approximation with errors propagated from the corresponding folding state. Application of the defined principles on two real Lactate dehydrogenase fed-batch refolding processes resulted in two individual sampling strategies. At reaction rates of up to 0.58 g h−1 description of the ongoing dynamics could be done accurately with high sampling intervals during the first few hours of processing as shown by comparison to a noise free simulation. The proposed method has the potential to be transferred to other proteins and to facilitate the development of model-based monitoring & control strategies.

Degradation-Suppressed Cocoonase for Investigating the Propeptide-Mediated Activation Mechanism.

Cocoonase is folded in the form of a zymogen precursor protein (prococoonase) with the assistance of the propeptide region. To investigate the role of the propeptide sequence on the disulfide-coupled folding of cocoonase and prococoonase, the amino acid residues at the degradation sites during the refolding and auto-processing reactions were determined by mass spectrometric analyses and were mutated to suppress the numerous degradation reactions that occur during the reactions. In addition, the Lys8 residue at the propeptide region was also mutated to estimate whether the entire sequence is absolutely required for the activation of cocoonase. Finally, a degradation-suppressed [K8D,K63G,K131G,K133A]-proCCN protein was prepared and was found to refold readily without significant degradation. The results of an enzyme assay using casein or Bz-Arg-OEt suggested that the mutations had no significant effect on either the enzyme activity or the protein conformation. Thus, we, herein, provide the non-degradative cocoonase protein to investigate the propeptide-mediated protein folding of the molecule. We also examined the catalytic residues using the degradation-suppressed cocoonase. The point mutations at the putative catalytic residues in cocoonase resulted in the loss of catalytic activity without any secondary structural changes, indicating that the mutated residues play a role in the catalytic activity of this enzyme.

Homemade plasmid Miniprep solutions for affordable research in low-fund laboratories

As a consequence of Covid-19 pandemic, the basic lab consumables are in shortage, especially in the low-income countries. Thus, the main objective of the present study is to develop and evaluate homemade solution to isolate plasmid. To pursue this objective, RNase A was overexpressed in Bl21 DE3 cells (E. coli strain) and prepared as crude refolding reaction with proper activity. Also, lysis buffers, neutralization buffer, and washing buffers were prepared. The homemade miniprep kit showed successful isolation of the px48SpCas9 plasmid. The prepared plasmid purity was enough to be used successfully in PCR amplification. In addition, to get extra benefits from this study, seven primers were designed to match the plasmid backbone to produce DNA ladder (100–1500 bp). In conclusion, we were able to have attainable working solutions for plasmid miniprep and DNA ladder.

The propeptide sequence assists the correct folding required for the enzymatic activity of cocoonase

Cocoonase, a protein that is produced by the silkworm (Bombyx mori), is thought to specifically digest the sericin protein of the cocoon and has a high homology with trypsin. Similar to trypsin, cocoonase is folded as an inactive precursor protein which is activated by releasing the propeptide moiety. However, the mechanism responsible for the activation of its catalytic structure has not yet been determined in detail. Therefore, to investigate the activation and folding mechanism of cocoonase, recombinant cocoonase (CCN) and prococoonase (proCCN) were over-expressed in E. coli cells.Both recombinant proteins (proCCN and CCN) were expressed as inclusion bodies in E. coli cells and their folding was examined under several sets of conditions. After the refolding reactions, both of the recombinant proteins were present as the oxidized soluble forms. The proCCN protein was then auto-processed to release the propeptide region for activation. Interestingly, the CCN (CCN∗) derived from the refolded proCCN showed a much stronger protease activity than the refolded CCN from the reduced CCN in a protease assay using Bz-Arg-OEt as a substrate. In addition, the secondary structure of the refolded CCN protein was similar to that of the CCN∗ protein, as evidenced by CD measurements. These results suggest that the CCN protein becomes trapped in a molten globule-like state without the assistance of the propeptide region during the folding process. We therefore conclude that the propeptide region of CCN kinetically accelerates the folding of CCN to adopt the correct conformation of cocoonase at the final step of the folding pathway.

Binding of 30S Ribosome Induces Single-stranded Conformation Within and Downstream of the Expression Platform in a Translational Riboswitch

Translational riboswitches are bacterial gene regulatory elements found in the 5′-untranslated region of mRNAs. They operate through a conformational refolding reaction that is triggered by a concentration change of a modulating small molecular ligand. The translation initiation region (TIR) is either released from or incorporated into base pairing interactions through the conformational switch. Hence, initiation of translation is regulated by the accessibility of the Shine-Dalgarno sequence and start codon. Interaction with the 30S ribosome is indispensable for the structural switch between functional OFF and ON states. However, on a molecular level it is still not fully resolved how the ribosome is accommodated near or at the translation initiation region in the context of translational riboswitches. The standby model of translation initiation postulates a binding site where the mRNA enters the ribosome and where it resides until the initiation site becomes unstructured and accessible. We here investigated the adenine-sensing riboswitch from Vibrio vulnificus. By application of a 19F labelling strategy for NMR spectroscopy that utilizes ligation techniques to synthesize differentially 19F labelled riboswitch molecules we show that nucleotides directly downstream of the riboswitch domain are first involved in productive interaction with the 30S ribosomal subunit. Upon the concerted action of ligand and the ribosomal protein rS1 the TIR becomes available and subsequently the 30S ribosome can slide towards the TIR. It will be interesting to see whether this is a general feature in translational riboswitches or if riboswitches exist where this region is structured and represent yet another layer of regulation.

The RNA chaperone StpA enables fast RNA refolding by destabilization of mutually exclusive base pairs within competing secondary structure elements.

In bacteria RNA gene regulatory elements refold dependent on environmental clues between two or more long-lived conformational states each associated with a distinct regulatory state. The refolding kinetics are strongly temperature-dependent and especially at lower temperatures they reach timescales that are biologically not accessible. To overcome this problem, RNA chaperones have evolved. However, the precise molecular mechanism of how these proteins accelerate RNA refolding reactions remains enigmatic. Here we show how the RNA chaperone StpA of Escherichia coli leads to an acceleration of a bistable RNA’s refolding kinetics through the selective destabilization of key base pairing interactions. We find in laser assisted real-time NMR experiments on photocaged bistable RNAs that the RNA chaperone leads to a two-fold increase in refolding rates at low temperatures due to reduced stability of ground state conformations. Further, we can show that upon interaction with StpA, base pairing interactions in the bistable RNA are modulated to favor refolding through the dominant pseudoknotted transition pathway. Our results shed light on the molecular mechanism of the interaction between RNA chaperones and bistable RNAs and are the first step into a functional classification of chaperones dependent on their biophysical mode of operation.

Application of 2,2′‐dipyridyl disulfide‐mediated thiazolidine ring‐opening reaction to glycoprotein synthesis: Total chemical synthesis of evasin‐3

Thiazolidine ring-opening reaction is one of the key steps in protein chemical synthesis via sequential native chemical ligation strategy. We recently developed a novel thiazolidine ring-opening reaction with 2,2'-dipyridyl disulfide (DPDS). In order to investigate the applicability of this reaction to glycoprotein synthesis, we synthesized evasin-3, a cysteine-rich glycoprotein with chemokine-binding ability originally found in tick saliva. The sequence of evasin-3 was divided into three segments, and these segments were separately synthesized with the ordinary solid-phase peptide synthesis method. After the first ligation of middle and C-terminal segments, thiazolidine used as a protecting group of Cys residue at the N-terminus of the middle segment was converted to Cys with DPDS. In this thiazolidine ring-opening reaction, DPDS treatment did not affect the N-linked glycan moiety. After the second ligation with the N-terminal segment and the refolding reaction, evasin-3 could be obtained in good yield. The synthetic evasin-3 showed the binding ability specifically to CXCL chemokines. These results clearly indicate that this DPDS method is useful for glycoprotein synthesis.

Multivariate Optimization of the Refolding Process of an Incorrectly Folded Fc-Fusion Protein in a Cell Culture Broth.

Protein misfolding is a common problem in large-scale production of recombinant proteins, which can significantly reduce the yield of the process. In this work, we aimed at treating a cell culture broth containing high levels (>45%) of incorrectly folded Fc-fusion proteins by a simple redox buffer system in order to increase the proportion of the protein with correct conformation. Multi-variable process optimization was firstly conducted at a small scale (25 mL), employing an experimental design methodology. After identifying the key variables using a resolution IV Fractional Factorial Design (FFD), the process was then optimized by the Central Composite Design (CCD). The optimal conditions for the refolding reaction were 340 mM Tris-base, 6.0 mM L-cysteine, 0.5 mM L-cystine, a buffer pH of 9.0, a reaction temperature of 8.5ºC and a reaction time of 24 h. Based on the treatment conditions obtained at a small scale, the process was further scaled up to 4500- L. The misfolded content was always less than 20%. The reaction can proceed well in the absence of chemical additives, such as chaotropic agents, aggregation suppressors, stabilizers and chelators. The refolding process increases the fraction of active protein in the original broth reducing the burden on downstream purification steps markedly.

Enzyme Activation Mechanism of Cocoonase

Cocoonase, a protein produced by the silkworm (Bombyx mori), consists of a pro-peptide and a mature region with three disulfide bonds. Cocoonase is known to specifically digest the sericin protein which is a coating protein of the fibroin of silkworm cocoons. However, the specificity of the protease activity and the mechanism responsible for its activation related to folding have not yet been determined in detail although its primary structure shows a high homology to that of trypsin. Therefore, to further study the activation mechanism and biological activity of cocoonase, we prepared recombinant cocoonase using an E. coli expression system, and examined its folding and enzymatic activity.For this purpose, a recombinant pro-cocoonase (pro-CCN), cocoonase (CCN), and its mutant proteins were expressed using the T7 expression system in E. coli cells. The recombinant proteins were expressed in the forms of inclusion bodies. Therefore, the refolding reaction was carried out in the presence of 2 mM GSH/1 mM GSSG under several different conditions after purification. In general, pro-CCN was treated with trypsin to eliminate the pro-peptide region for its enzyme activation. To avoid contamination by the trypsin activity in the final product, the refolded pro-CCN was further processed to the mature form, CCN, by the autolysis of the pro-peptide region in the absence of trypsin at several temperatures. The enzyme activity of the recombinant CCN derived from pro-CCN was then estimated, either by a casein assay or the hydrolysis of Bz-Arg-OEt as a substrate. In addition, to eliminate the non-specific degradation of CCN during the refolding reaction, a protease-resistant mutant was also prepared and its folding examined.

Intra-Molecular Chaperone Mediated Folding of a Peptide Hormone in Molecular Evolution

Uroguanylin, an endogenous ligand of guanylyl cyclase C, consists of 16 amino acid residues and contains two intra-molecular disulfide bonds. The folding of uroguanylin is regulated by the assistance of the pro-peptide region that functions as an intra-molecular chaperone. Our previous studies suggested that a mis-bridged disulfide isomer of prouroguanylin is formed at an early stage of the refolding reaction and the molecule is then converted to the native conformation. Importantly, this folding mechanism involves a change in the secondary structure from an α-helix to a β-sheet structure at the processing site and at the mature region, respectively. However, eel-prouroguanylin possesses three Pro residues at the processing site, which inhibits α-helix formation during folding. To investigate the folding mechanism and quality control during molecular evolution, a cassette mutation was incorporated into eel and human prouroguanylin and its folding examined.The cassette mutants were expressed in E. coli cells using the T7-expression system, and produced as inclusion bodies. Therefore, the proteins were solubilized in 8 M urea containing dithiothreitol and purified by a reversed-phase high performance liquid chromatography as the reduced forms. The oxidative refolding reactions of the cassette mutants were then examined under the conditions of kinetic or thermodynamic control. The folding mechanism of the mutants and wild type prouroguanylins were compared among several species of prourogaunylin and the findings were evaluated from the standpoint of molecular evolution. The results are discussed in this paper.

Molecular and Biological Characterization of a Prepared Recombinant Human Interferon Alpha 2b Isoform.

Recombinant human interferon alpha2b (rhIFN-α2b) protein is FDA approved for treatment of many tumors and viral diseases. A rhIFN-α2b isoform has been produced and purified from the refolding reaction using high-resolution anion ion exchange chromatography. This isoform has a proper MW (19kDa) and high purity and homogeneity. The conservation of native linear and conformational epitopes in this isoform was immunologically confirmed by Western blot and ELISA. Mass spectrometry assessment of its intact mass showed average mass (19,337Da) equivalent to that of the expressed rhIFN-α2b protein without any chemical modification and without the first methionine. Peptide mapping of rhIFN-α2b through tryptic digestion of reductive/alkylated protein using urea as a denaturing agent gave the best pattern. The rhIFN-α2b had a high specific antiviral activity (2.5 × 108 ± 1.1 × 108IU/mg protein). In vivo clearance study of rhIFN-α2b in female SD rats (500μg/kg, intramuscularly) revealed rapid clearance (elimination half-life 0.54h with a maximum plasma concentration of 33,792pg/ml) compared with the commercial rhIFN-α2 (elimination half-life 0.75-0.96h). In conclusion, the prepared rhIFN-α2b isoform has high purity, homogeneity, native like chemical and structural composition, high antiviral activity, and proper biological stability, which reduce its immunogenicity and raise its therapeutic efficiency.

Effects of osmolytes on the refolding of recombinant Pelodiscus sinensis brain-type creatine kinase

Abstract The protective effects of osmolytes on the renaturation of Pelodiscus sinensis brain-type creatine kinase (P-CKB) were determined in this study. The P-CKB gene was cloned and was heterologously expressed in Escherichia coli BL21 (DE3). The purified recombinant protein was subjected to 6 M urea denaturation and further applied with six different osmolytes (glycine, proline, sorbitol, DMSO, betaine, and trehalose). Our results demonstrated that the addition of glycine or DMSO could contribute toward the reactivation of unfolded P-CKB and prevent aggregation. Interestingly, 25 mM glycine was found to be the best concentration to reactivate denatured P-CKB, while high-concentration glycine led to the opposite effect as monitored by a red shift in the intrinsic fluorescence spectra and the aggregation of protein, which probably could be attributed to an additional inactive protein complex that formed during the fast refolding reaction. Docking simulation results showed the osmolyte docking energies to be relatively low and clustering groups were spread on the surface of P-CKB, indicating that osmolytes directly protected the surface of P-CKB. The results in this study could provide a better understanding of structural and functional changes in P-CKB during refolding in addition to the role of osmolytes in heterothermic animals.

Folding Analyses of a de novo Designed Prouroguanylin

Peptide hormones are typically produced in the form of a prepro-peptide, precursor molecule, which is then processed into the biologically active mature form. However, the role of the pro-peptide region, especially its role in the correct folding which is absolutely required for biological activity, has not been explained in detail. Uroguanylin folds into its native conformation with the assistance of the pro-peptide region, which functions as an intra-molecular chaperone. We recently applied its chaperone function to construct a de novo designed peptide, which is able to fold into only a bioactive conformation via the fusion of the propeptide region of uroguanylin. In addition, our previous studies of the disulfide-coupled folding of a series of Gly mutants of prouroguanylin suggested that only a few amino acid residues of prouroguanylin showed critical effects in the formation of the native conformation. To further investigate the mechanism for the propeptide-mediated folding of peptide hormones, the several amino acid residues of the de novo fusion protein were mutated to Gly residues and the refolding reactions of these proteins were examined. The mutant proteins were prepared using an E. coli expression system. The mutant proteins were produced in the form of inclusion bodies and then solubilized in 8 M urea containing dithiothreitol. The reduced forms of the mutant proteins were purified by RP-HPLC and the structures confirmed by MALDI-TOF/MS analyses. The oxidative refolding reactions of the proteins were carried out in the presence or absence of glutathione. The results of our studies will be discussed in this paper.

Disulfide-Coupled Folding of Prouroguanylin on Molecular Evolution

The folding of uroguanylin is assisted by the pro-peptide region, which functions as an intra-molecular chaperone. Our previous study of the disulfide-coupled folding of prouroguanylin suggested that a mis-bridged disulfide isomer is produced at the early stage of folding and the molecule is then shifted to the native conformation, accompanied by a secondary structural change from an alpha-helix to a beta-sheet structure at the processing site and the C-terminal region, respectively. Thus, the necessity of a folding intermediate being produced suggests that the mechanism responsible for the chaperone-assisted folding of prouroguanylin includes quality control in constructing the native conformation expressly through the formation of a non-native disulfide isomer as the folding intermediate on molecular evolution. To validate this hypothesis, the folding of eel uroguanylin and prouroguanylin was compared to that for human uroguanylin and prouroguanylin, respectively. For this purpose, a series of N-terminal extended eel uroguanylin analogs was chemically synthesized. And a recombinant eel prouroguanylin was expressed as a thioredoxin-fused protein in E. coli cells because the expression efficiency of the recombinant eel prouroguanylin was quite low. The fusion protein was purified by Ni-affinity chromatography and treated with PreScission protease to release the eel prouroguanylin. The refolding reactions of eel uroguanylin analogs and eel prouroguanylin were then examined and the folding mechanism of eel prouroguanylin was compared to that for human prouroguanylin.

Kinetic stability and sequence/structure studies of urine-derived Bence-Jones proteins from multiple myeloma and light chain amyloidosis patients

It is now accepted that the ability of a protein to form amyloid fibrils could be associated both kinetic and thermodynamic protein folding parameters. A recent study from our laboratory using recombinant full-length (encompassing the variable and constant domain) immunoglobulin light chains found a strong kinetic control of the protein unfolding for these proteins. In this study, we are extending our analysis by using urine-derived Bence Jones proteins (BJPs) from five patients with light chain (AL) amyloidosis and four patients with multiple myeloma (MM).We observed lower stability in κ proteins compared to λ proteins (for both MM and AL proteins) in agreement with previous studies. The kinetic component of protein stability is not a universal feature of BJPs and the hysteresis observed during refolding reactions could be attributed to the inability of the protein to refold all domains.The most stable proteins exhibited 3-state unfolding transitions. While these proteins do not refold reversibly, partial refolding shows 2-state partial refolding transitions, suggesting that one of the domains (possibly the variable domain) does not refold completely. Sequences were aligned with their respective germlines and the location and nature of the mutations were analyzed. The location of the mutations were analyzed and compared with the stability and amyloidogenic properties for the proteins in this study, increasing our understanding of light chain unfolding and amyloidogenic potential.

Screening of Neem extracts for microbial anti-chaperone activity by employing in vitro enzyme refolding assay.

Microbial heat shock proteins(Hsps) play an important role in pathogenesis and development of resistance to existing drugs. New compounds that target microbial molecular chaperones have the potential of combating the challenge of anti-microbial resistance. The present study was aimed at assessing the employment of in vitro enzyme refolding assay to detect anti-chaperone activity of Neem (Azadirachta indica) extracts. Protein extracts of thermotolerant Escherichia coli cells were used as a source of Hsps or chaperones. Thermotolerance was found to be induced by pre-treating E. coli cells at 47°C before subjecting them to a lethal temperature of 55°C. This thermotolerance correlated with over-expression of specific proteins and reduced aggregation as evident from the SDS-PAGE profiles. Refolding assays of denatured enzymes exhibited 45% activity regain in presence of cell protein extracts containing chaperones compared to less than 5% regain in BSA negative controls. The chaperone activity was found to be ATP dependent. Addition of Neem extracts to refolding reaction mixtures distinctly reduced the activity regain (20%) in a dose dependent manner (500 and 1000ppm). The negative influence of plant extract on refolding of the enzyme in the presence of chaperones gives evidence to its anti-chaperone activity. We propose that the employment of in vitro enzyme refolding assays will help not only to analyze the activity of known and putative chaperones but also to screen natural compounds for anti-microbial-Hsp activity.

Substrate binding by the yeast Hsp110 nucleotide exchange factor and molecular chaperone Sse1 is not obligate for its biological activities.

The highly conserved heat shock protein 70 (Hsp70) is a ubiquitous molecular chaperone essential for maintaining cellular protein homeostasis. The related protein Hsp110 (Sse1/Sse2 in Saccharomyces cerevisiae) functions as a nucleotide exchange factor (NEF) to regulate the protein folding activity of Hsp70. Hsp110/Sse1 also can prevent protein aggregation in vitro via its substrate-binding domain (SBD), but the cellular roles of this "holdase" activity are poorly defined. We generated and characterized an Sse1 mutant that separates, for the first time, its nucleotide exchange and substrate-binding functions. Sse1sbd retains nucleotide-binding and nucleotide exchange activities while exhibiting severe deficiencies in chaperone holdase activity for unfolded polypeptides. In contrast, we observed no effect of the SBD mutation in reconstituted disaggregation or refolding reactions in vitro. In vivo, Sse1sbd successfully heterodimerized with the yeast cytosolic Hsp70s Ssa and Ssb and promoted normal growth, with the exception of sensitivity to prolonged heat but not other proteotoxic stress. Moreover, Sse1sbd was fully competent to support Hsp90-dependent signaling through heterologously expressed glucocorticoid receptor and degradation of a permanently misfolded protein, two previously defined roles for Sse1. We conclude that despite conservation among eukaryotic homologues, chaperone holdase activity is not an obligate function in the Hsp110 family.