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The Journal of Biological Chemistry Aug 2018Most protein folding studies until now focus on single domain or truncated proteins. Although great insights in the folding of such systems has been accumulated, very...
Most protein folding studies until now focus on single domain or truncated proteins. Although great insights in the folding of such systems has been accumulated, very little is known regarding the proteins containing multiple domains. It has been shown that the high stability of domains, in conjunction with inter-domain interactions, manifests as a frustrated energy landscape, causing complexity in the global folding pathway. However, multidomain proteins despite containing independently foldable, loosely cooperative sections can fold into native states with amazing speed and accuracy. To understand the complexity in mechanism, studies were conducted previously on the multidomain protein malate synthase G (MSG), an enzyme of the glyoxylate pathway with four distinct and adjacent domains. It was shown that the protein refolds to a functionally active intermediate state at a fast rate, which slowly produces the native state. Although experiments decoded the nature of the intermediate, a full description of the folding pathway was not elucidated. In this study, we use a battery of biophysical techniques to examine the protein's folding pathway. By using multiprobe kinetics studies and comparison with the equilibrium behavior of protein against urea, we demonstrate that the unfolded polypeptide undergoes conformational compaction to a misfolded intermediate within milliseconds of refolding. The misfolded product appears to be stabilized under moderate denaturant concentrations. Further folding of the protein produces a stable intermediate, which undergoes partial unfolding-assisted large segmental rearrangements to achieve the native state. This study reveals an evolved folding pathway of the multidomain protein MSG, which involves surpassing the multiple misfolding traps during refolding.
Topics: Crystallography, X-Ray; Escherichia coli; Kinetics; Malate Synthase; Models, Molecular; Protein Conformation; Protein Denaturation; Protein Folding; Protein Refolding; Thermodynamics
PubMed: 29959230
DOI: 10.1074/jbc.RA118.003903 -
Cell Chemical Biology Oct 2018Reactivation of mutant p53 has emerged as a promising approach for cancer therapy. Recent studies have identified several mutant p53-reactivating compounds that target...
Reactivation of mutant p53 has emerged as a promising approach for cancer therapy. Recent studies have identified several mutant p53-reactivating compounds that target thiol groups in mutant p53. Here we have investigated the relationship between thiol reactivity, p53 thermostabilization, mutant p53 refolding, mutant p53-dependent growth suppression, and induction of cell death. Analysis of the National Cancer Institute database revealed that Michael acceptors show the highest selectivity for mutant p53-expressing cells among analyzed thiol-reactive compounds. Further experimental testing demonstrated that Michael acceptors, aldehydes, imines, and primary alcohols can promote thermodynamic stabilization of mutant p53. Moreover, mild thiol reactivity, often coupled with combined chemical functional groups, such as in imines, aldehydes, and primary alcohols, can stimulate mutant p53 refolding. However, strong electrophile activity was associated with cellular toxicity. Our findings may open possibilities for rational design of novel potent and selective mutant p53-reactivating compounds.
Topics: Antineoplastic Agents; Apoptosis; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Humans; Models, Molecular; Neoplasms; Point Mutation; Protein Refolding; Protein Stability; Reactive Oxygen Species; Sulfhydryl Compounds; Tumor Suppressor Protein p53
PubMed: 30057300
DOI: 10.1016/j.chembiol.2018.06.013 -
Protein Expression and Purification Apr 2012Bone morphogenetic proteins (BMPs) are secreted protein ligands that control numerous biological processes, such as cell differentiation and cell proliferation. Ligands...
Bone morphogenetic proteins (BMPs) are secreted protein ligands that control numerous biological processes, such as cell differentiation and cell proliferation. Ligands are regulated by a large number of structurally diverse extracellular antagonists. PRDC or protein related to DAN and cerberus is a BMP antagonist of the DAN family, which is defined by a conserved pattern of cysteine residues that form a ring structure. Here we present the expression and purification of recombinant mouse PRDC (mPRDC) from bacterial (Escherichia coli) inclusion bodies through oxidative refolding. Functional mPRDC was isolated from a nonfunctional component through reverse phase chromatography and shown to inhibit BMP2 and BMP4 in a cell-based luciferase reporter assay. Recombinant mPRDC also bound directly to BMP2, BMP4 and BMP7, but not activin A. Furthermore, circular dichroism indicated that mPRDC is folded and contains a higher than anticipated helical content for a DAN family member protein.
Topics: Animals; Bone Morphogenetic Proteins; Cell Line; Chromatography, Reverse-Phase; Cytokines; Gene Expression; Genes, Reporter; Hydrophobic and Hydrophilic Interactions; Inclusion Bodies; Luciferases; Mice; Protein Binding; Protein Refolding; Protein Structure, Secondary; Proteins; Recombinant Proteins; Signal Transduction; Transforming Growth Factor beta
PubMed: 22381466
DOI: 10.1016/j.pep.2012.02.010 -
Tracking unfolding and refolding reactions of single proteins using atomic force microscopy methods.Methods (San Diego, Calif.) Apr 2013During the last two decades single-molecule manipulation techniques such as atomic force microscopy (AFM) has risen to prominence through their unique capacity to...
During the last two decades single-molecule manipulation techniques such as atomic force microscopy (AFM) has risen to prominence through their unique capacity to provide fundamental information on the structure and function of biomolecules. Here we describe the use of single-molecule AFM to track protein unfolding and refolding pathways, enzymatic catalysis and the effects of osmolytes and chaperones on protein stability and folding. We will outline the principles of operation for two different AFM pulling techniques: length clamp and force-clamp and discuss prominent applications. We provide protocols for the construction of polyproteins which are amenable for AFM experiments, the preparation of different coverslips, choice and calibration of AFM cantilevers. We also discuss the selection criteria for AFM recordings, the calibration of AFM cantilevers, protein sample preparations and analysis of the obtained data.
Topics: Buffers; Calibration; Computer Simulation; Humans; Immobilized Proteins; Mechanical Phenomena; Microscopy, Atomic Force; Models, Molecular; Osmolar Concentration; Polyproteins; Protein Refolding; Protein Unfolding
PubMed: 23523554
DOI: 10.1016/j.ymeth.2013.03.010 -
Journal of the American Chemical Society Nov 2009Semisynthetic green fluorescent proteins (GFPs) can be prepared by producing truncated GFPs recombinantly and assembling them with synthetic beta-strands of GFP. The...
Semisynthetic green fluorescent proteins (GFPs) can be prepared by producing truncated GFPs recombinantly and assembling them with synthetic beta-strands of GFP. The yield from expressing the truncated GFPs is low, and the chromophore is either partially formed or not formed. An alternative method is presented in which full-length proteins are produced recombinantly with a protease site inserted between the structural element to be removed and the rest of the protein. The native peptide can then be replaced by cutting the protease site with trypsin, denaturing in guanidine hydrochloride to disrupt the complex, separating the native peptide from the rest of the protein by size exclusion, and refolding the protein in the presence of a synthetic peptide. We show that this method allows for removal and replacement of the interior chromophore containing helix and that the GFP barrel is capable of inducing chromophore formation in a synthetic interior helix.
Topics: Green Fluorescent Proteins; Peptide Hydrolases; Protein Conformation; Protein Denaturation; Protein Renaturation; Recombinant Proteins; Recombination, Genetic; Trypsin
PubMed: 19839621
DOI: 10.1021/ja906303f -
Scientific Reports Feb 2019Molecular chaperones play an important role in cellular protein-folding assistance and aggregation inhibition. As a different but complementary model, we previously...
Molecular chaperones play an important role in cellular protein-folding assistance and aggregation inhibition. As a different but complementary model, we previously proposed that, in general, soluble cellular macromolecules with large excluded volume and surface charges exhibit intrinsic chaperone activity to prevent aggregation of their connected polypeptides irrespective of the connection type, thereby contributing to efficient protein folding. As a proof of concept, we here demonstrated that a model recombinant protein with a specific sequence-binding domain robustly exerted chaperone activity toward various proteins harbouring a short recognition tag of 7 residues in Escherichia coli. The chaperone activity of this protein was comparable to that of representative E. coli chaperones in vivo. Furthermore, in vitro refolding experiments confirmed the in vivo results. Our findings reveal that a soluble protein exhibits the intrinsic chaperone activity to prevent off-pathway aggregation of its interacting proteins, leading to more productive folding while allowing them to fold according to their intrinsic folding pathways. This study gives new insights into the plausible chaperoning role of soluble cellular macromolecules in terms of aggregation inhibition and indirect folding assistance.
Topics: Binding Sites; Escherichia coli; Escherichia coli Proteins; Molecular Chaperones; Protein Aggregates; Protein Binding; Protein Folding; Protein Refolding; Recombinant Proteins; Solubility
PubMed: 30804538
DOI: 10.1038/s41598-019-39158-6 -
Protein Science : a Publication of the... Nov 2010Refolding of proteins from solubilized inclusion bodies still represents a major challenge for many recombinantly expressed proteins and often constitutes a major...
Refolding of proteins from solubilized inclusion bodies still represents a major challenge for many recombinantly expressed proteins and often constitutes a major bottleneck. As in vitro refolding is a complex reaction with a variety of critical parameters, suitable refolding conditions are typically derived empirically in extensive screening experiments. Here, we introduce a new strategy that combines screening and optimization of refolding yields with a genetic algorithm (GA). The experimental setup was designed to achieve a robust and universal method that should allow optimizing the folding of a variety of proteins with the same routine procedure guided by the GA. In the screen, we incorporated a large number of common refolding additives and conditions. Using this design, the refolding of four structurally and functionally different model proteins was optimized experimentally, achieving 74-100% refolding yield for all of them. Interestingly, our results show that this new strategy provides optimum conditions not only for refolding but also for the activity of the native enzyme. It is designed to be generally applicable and seems to be eligible for all enzymes.
Topics: Algorithms; Computational Biology; Disulfides; Models, Genetic; Models, Molecular; Protein Refolding; Recombinant Proteins; Stochastic Processes
PubMed: 20799347
DOI: 10.1002/pro.488 -
Current Genetics Oct 2019The Heat Shock Protein 70s (Hsp70s) are an essential family of proteins involved in folding of new proteins and triaging of damaged proteins for proteasomal-mediated... (Review)
Review
The Heat Shock Protein 70s (Hsp70s) are an essential family of proteins involved in folding of new proteins and triaging of damaged proteins for proteasomal-mediated degradation. They are highly conserved in all organisms, with each organism possessing multiple highly similar Hsp70 variants (isoforms). These isoforms have been previously thought to be identical in function differing only in their spatio-temporal expression pattern. The model organism Saccharomyces cerevisiae (baker's yeast) expresses four Hsp70 isoforms Ssa1, 2, 3 and 4. Here, we review recent findings that suggest that despite their similarity, Ssa isoforms may have unique cellular functions.
Topics: Adenosine Triphosphatases; Cytosol; Fungal Proteins; Gene Expression Regulation, Fungal; HSP70 Heat-Shock Proteins; Protein Aggregates; Protein Binding; Protein Isoforms; Protein Processing, Post-Translational; Protein Refolding; Signal Transduction; Structure-Activity Relationship; Yeasts
PubMed: 31020385
DOI: 10.1007/s00294-019-00978-8 -
PloS One 2014Some years ago, we showed that thermo-chemically denatured, partially-unfolded forms of Pyrococcus furiosus triosephosphateisomerase (PfuTIM) display cold-denaturation...
Some years ago, we showed that thermo-chemically denatured, partially-unfolded forms of Pyrococcus furiosus triosephosphateisomerase (PfuTIM) display cold-denaturation upon cooling, and heat-renaturation upon reheating, in proportion with the extent of initial partial unfolding achieved. This was the first time that cold-denaturation was demonstrated for a hyperthermophile protein, following unlocking of surface salt bridges. Here, we describe the behavior of another hyperthermophile protein, the small, monomeric, 53 residues-long rubredoxin from Pyrococcus furiosus (PfRd), which is one of the most thermostable proteins known to man. Like PfuTIM, PfRd too displays cold-denaturation after initial thermo-chemical perturbation, however, with two differences: (i) PfRd requires considerably higher temperatures as well as higher concentrations of guanidium hydrochloride (Gdm.HCl) than PfuTIM; (ii) PfRd's cold-denaturation behavior during cooling after thermo-chemical perturbation is incompletely reversible, unlike PfuTIM's, which was clearly reversible (from each different conformation generated). Differential cold-denaturation treatments allow PfRd to access multiple partially-unfolded states, each of which is clearly highly kinetically-stable. We refer to these as 'Trishanku' unfolding intermediates (or TUIs). Fascinatingly, refolding of TUIs through removal of Gdm.HCl generates multiple partially-refolded, monomeric, kinetically-trapped, non-native 'Trishanku' refolding intermediates (or TRIs), which differ from each other and from native PfRd and TUIs, in structural content and susceptibility to proteolysis. We find that the occurrence of cold denaturation and observations of TUI and TRI states is contingent on the oxidation status of iron, with redox agents managing to modulate the molecule's behavior upon gaining access to PfRd's iron atom. Mass spectrometric examination provides no evidence of the formation of disulfide bonds, but other experiments suggest that the oxidation status of iron (and its extent of burial) together determine whether or not PfRd shows cold denaturation, and also whether redox agents are able to modulate its behavior.
Topics: Amino Acid Sequence; Archaeal Proteins; Base Sequence; Circular Dichroism; Cold Temperature; Electrophoresis, Polyacrylamide Gel; Hot Temperature; Models, Molecular; Molecular Sequence Data; Oxidation-Reduction; Protein Conformation; Protein Denaturation; Protein Refolding; Protein Stability; Pyrococcus furiosus; Rubredoxins; Sequence Analysis, DNA; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Thermodynamics
PubMed: 24603413
DOI: 10.1371/journal.pone.0080014 -
Microbial Cell Factories Aug 2022Native-like secondary structures and biological activity have been described for proteins in inclusion bodies (IBs). Tertiary structure analysis, however, is hampered...
BACKGROUND
Native-like secondary structures and biological activity have been described for proteins in inclusion bodies (IBs). Tertiary structure analysis, however, is hampered due to the necessity of mild solubilization conditions. Denaturing reagents used for IBs solubilization generally lead to the loss of these structures and to consequent reaggregation due to intermolecular interactions among exposed hydrophobic domains after removal of the solubilization reagent. The use of mild, non-denaturing solubilization processes that maintain existing structures could allow tertiary structure analysis and increase the efficiency of refolding.
RESULTS
In this study we use a variety of biophysical methods to analyze protein structure in human growth hormone IBs (hGH-IBs). hGH-IBs present native-like secondary and tertiary structures, as shown by far and near-UV CD analysis. hGH-IBs present similar λ intrinsic Trp fluorescence to the native protein (334 nm), indicative of a native-like tertiary structure. Similar fluorescence behavior was also obtained for hGH solubilized from IBs and native hGH at pH 10.0 and 2.5 kbar and after decompression. hGH-IBs expressed in E. coli were extracted to high yield and purity (95%) and solubilized using non-denaturing conditions [2.4 kbar, 0.25 M arginine (pH 10), 10 mM DTT]. After decompression, the protein was incubated at pH 7.4 in the presence of the glutathione-oxidized glutathione (GSH-GSSG) pair which led to intramolecular disulfide bond formation and refolded hGH (81% yield).
CONCLUSIONS
We have shown that hGH-IBs present native-like secondary and tertiary structures and that non-denaturing methods that aim to preserve them can lead to high yields of refolded protein. It is likely that the refolding process described can be extended to different proteins and may be particularly useful to reduce the pH required for alkaline solubilization.
Topics: Humans; Escherichia coli; Human Growth Hormone; Inclusion Bodies; Protein Refolding; Protein Structure, Secondary; Protein Structure, Tertiary; Recombinant Proteins; Solubility
PubMed: 35978337
DOI: 10.1186/s12934-022-01887-1