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Journal of the American Chemical Society Mar 2014Density is an easily adjusted variable in molecular dynamics (MD) simulations. Thus, pressure-jump (P-jump)-induced protein refolding, if it could be made fast enough,...
Density is an easily adjusted variable in molecular dynamics (MD) simulations. Thus, pressure-jump (P-jump)-induced protein refolding, if it could be made fast enough, would be ideally suited for comparison with MD. Although pressure denaturation perturbs secondary structure less than temperature denaturation, protein refolding after a fast P-jump is not necessarily faster than that after a temperature jump. Recent P-jump refolding experiments on the helix bundle λ-repressor have shown evidence of a <3 μs burst phase, but also of a ~1.5 ms "slow" phase of refolding, attributed to non-native helical structure frustrating microsecond refolding. Here we show that a λ-repressor mutant is nonetheless capable of refolding in a single explicit solvent MD trajectory in about 19 μs, indicating that the burst phase observed in experiments on the same mutant could produce native protein. The simulation reveals that after about 18.5 μs of conformational sampling, the productive structural rearrangement to the native state does not occur in a single swift step but is spread out over a brief series of helix and loop rearrangements that take about 0.9 μs. Our results support the molecular time scale inferred for λ-repressor from near-downhill folding experiments, where transition-state population can be seen experimentally, and also agrees with the transition-state transit time observed in slower folding proteins by single-molecule spectroscopy.
Topics: Molecular Dynamics Simulation; Pressure; Protein Refolding; Proteins
PubMed: 24437525
DOI: 10.1021/ja412639u -
PloS One 2022SARS-CoV-2 Nucleocapsid (N) is the most abundant viral protein expressed in host samples and is an important antigen for diagnosis. N is a 45 kDa protein that does not...
SARS-CoV-2 Nucleocapsid (N) is the most abundant viral protein expressed in host samples and is an important antigen for diagnosis. N is a 45 kDa protein that does not present disulfide bonds. Intending to avoid non-specific binding of SARS-CoV-2 N to antibodies from patients who previously had different coronaviruses, a 35 kDa fragment of N was expressed without a conserved motif in E. coli as inclusion bodies (N122-419-IB). Culture media and IB washing conditions were chosen to obtain N122-419-IB with high yield (370 mg/L bacterial culture) and protein purity (90%). High pressure solubilizes protein aggregates by weakening hydrophobic and ionic interactions and alkaline pH promotes solubilization by electrostatic repulsion. The association of pH 9.0 and 2.4 kbar promoted efficient solubilization of N122-419-IB without loss of native-like tertiary structure that N presents in IB. N122-419 was refolded with a yield of 85% (326 mg/L culture) and 95% purity. The refolding process takes only 2 hours and the protein is ready for use after pH adjustment, avoiding the necessity of dialysis or purification. Antibody binding of COVID-19-positive patients sera to N122-419 was confirmed by Western blotting. ELISA using N122-419 is effective in distinguishing between sera presenting antibodies against SARS-CoV-2 from those who do not. To the best of our knowledge, the proposed condition for IB solubilization is one of the mildest described. It is possible that the refolding process can be extended to a wide range of proteins with high yields and purity, even those that are sensible to very alkaline pH.
Topics: Antibodies, Viral; Antigens, Viral; COVID-19; Coronavirus Nucleocapsid Proteins; Enzyme-Linked Immunosorbent Assay; Escherichia coli; Humans; Hydrogen-Ion Concentration; Hydrostatic Pressure; Immunoglobulin G; Inclusion Bodies; Phosphoproteins; Protein Refolding; Protein Structure, Tertiary; Recombinant Proteins; SARS-CoV-2; Solubility
PubMed: 35113919
DOI: 10.1371/journal.pone.0262591 -
The FEBS Journal May 2017Tuberculosis, a contagious disease of infectious origin is currently a major cause of deaths worldwide. Mycobacterium indicus pranii (MIP), a saprophytic nonpathogen and... (Comparative Study)
Comparative Study
Tuberculosis, a contagious disease of infectious origin is currently a major cause of deaths worldwide. Mycobacterium indicus pranii (MIP), a saprophytic nonpathogen and a potent immunomodulator is currently being investigated as an intervention against tuberculosis along with many other diseases with positive outcome. The apparent paradox of multiple chaperones in mycobacterial species and enigma about the cellular functions of the client proteins of these chaperones need to be explored. Chaperones are the known immunomodulators; thus, there is need to exploit the proteome of MIP for identification and characterization of putative chaperones. One of the immunogenic proteins, MIP_05962 is a member of heat shock protein (HSP) 20 family due to the presence of α-crystallin domain, and has amino acid similarity with Mycobacterium lepraeHSP18 protein. The diverse functions of M. lepraeHSP18 in stress conditions implicate MIP_05962 as an important protein that needs to be explored. Biophysical and biochemical characterization of the said protein proved it to be a chaperone. The observations of aggregation prevention and refolding of substrate proteins in the presence of MIP_05962 along with interaction with non-native proteins, surface hydrophobicity, formation of large oligomers, in-vivo thermal rescue of Escherichia coli expressing MIP_05962, enhancing solubility of insoluble protein maltodextrin glucosidase (MalZ) under in-vivo conditions, and thermal stability and reversibility confirmed MIP_05962 as a molecular chaperone.
Topics: Amino Acid Sequence; Bacterial Proteins; Cell Survival; Citrate (si)-Synthase; Enzyme Stability; Escherichia coli; Escherichia coli Proteins; Glycoside Hydrolases; HSP20 Heat-Shock Proteins; Heat-Shock Response; Hot Temperature; Hydrophobic and Hydrophilic Interactions; Molecular Chaperones; Mycobacterium avium Complex; Protein Aggregates; Protein Folding; Protein Interaction Domains and Motifs; Protein Refolding; Recombinant Fusion Proteins; Sequence Alignment; Sequence Homology, Amino Acid; Solubility
PubMed: 28296245
DOI: 10.1111/febs.14057 -
The Journal of Biological Chemistry Apr 2005Protein disulfide isomerase (PDI) functions as an isomerase to catalyze thiol:disulfide exchange, as a chaperone to assist protein folding, and as a subunit of...
Protein disulfide isomerase (PDI) functions as an isomerase to catalyze thiol:disulfide exchange, as a chaperone to assist protein folding, and as a subunit of prolyl-4-hydroxylase and microsomal triglyceride transfer protein. At a lower concentration of 0.2 microm, PDI facilitated the aggregation of unfolded rabbit muscle creatine kinase (CK) and exhibited anti-chaperone activity, which was shown to be mainly due to the hydrophobic interactions between PDI and CK and was independent of the cross-linking of disulfide bonds. At concentrations above 1 microm, PDI acted as a protector against aggregation but an inhibitor of reactivation during CK refolding. The inhibition effect of PDI on CK reactivation was further characterized as due to the formation of PDI-CK complexes through intermolecular disulfide bonds, a process involving Cys-36 and Cys-295 of PDI. Two disulfide-linked complexes containing both PDI and CK were obtained, and the large, soluble aggregates around 400 kDa were composed of 1 molecule of tetrameric PDI and 2 molecules of inactive intermediate dimeric CK, whereas the smaller one, around 200 kDa, was formed by 1 dimeric PDI and 1 dimeric CK. To our knowledge this is the first study revealing that PDI could switch its conformation from dimer to tetramer in its functions as a foldase. According to the observations in this research and our previous study of the folding pathways of CK, a working model was proposed for the molecular mechanism of CK refolding catalyzed by PDI.
Topics: Animals; Creatine Kinase; Dithiothreitol; Guanidine; Humans; Isoenzymes; Models, Theoretical; Molecular Chaperones; Protein Denaturation; Protein Disulfide-Isomerases; Protein Folding; Protein Renaturation; Protein Structure, Quaternary; Rabbits
PubMed: 15695804
DOI: 10.1074/jbc.M413882200 -
Protein Expression and Purification Jul 2021Sensitive and specific serology tests are essential for epidemiological and public health studies of COVID-19 and for vaccine efficacy testing. The presence of...
Sensitive and specific serology tests are essential for epidemiological and public health studies of COVID-19 and for vaccine efficacy testing. The presence of antibodies to SARS-CoV-2 surface glycoprotein (Spike) and, specifically, its receptor-binding domain (RBD) correlates with inhibition of SARS-CoV-2 binding to the cellular receptor and viral entry into the cells. Serology tests that detect antibodies targeting RBD have high potential to predict COVID-19 immunity and to accurately determine the extent of the vaccine-induced immune response. Cost-effective methods of expression and purification of Spike and its fragments that preserve antigenic properties are essential for development of such tests. Here we describe a method of production of His6-tagged S319-640 fragment containing RBD in E. coli. It includes expression of the fragment, solubilization of inclusion bodies, and on-the-column refolding. The antigenic properties of the resulting product are similar, but not identical to the RBD-containing fragment expressed in human cells.
Topics: Binding Sites; COVID-19; Cloning, Molecular; Escherichia coli; Gene Expression; Humans; Peptide Fragments; Protein Domains; Protein Refolding; SARS-CoV-2; Solubility; Spike Glycoprotein, Coronavirus
PubMed: 33667651
DOI: 10.1016/j.pep.2021.105861 -
Croatian Medical Journal Apr 2020Health can be defined as a harmony, or homeostasis, of the activities of thousands of different proteins, whereas aging and diseases result from their disharmony... (Review)
Review
Health can be defined as a harmony, or homeostasis, of the activities of thousands of different proteins, whereas aging and diseases result from their disharmony manifested at the levels of cells and tissues. Such disharmony is caused primarily by dysfunction and toxicity of misfolded proteins damaged by oxidation. This is an overview of key data that inspired new concepts allowing interpretation and integration of the scientific literature on aging and age-related diseases. These concepts suggest strategies for prevention and attenuation of age-related degenerative and malignant diseases mimicking the life of super-centenarians.
Topics: Aged; Aging; DNA Damage; Humans; Neoplasms; Neurodegenerative Diseases; Oxidation-Reduction; Protein Refolding; Proteins; Proteolysis
PubMed: 32378382
DOI: 10.3325/cmj.2020.61.159 -
Science Advances May 2023Ring-forming AAA chaperones solubilize protein aggregates and protect organisms from proteostatic stress. In metazoans, the AAA chaperone Skd3 in the mitochondrial...
Ring-forming AAA chaperones solubilize protein aggregates and protect organisms from proteostatic stress. In metazoans, the AAA chaperone Skd3 in the mitochondrial intermembrane space (IMS) is critical for human health and efficiently refolds aggregated proteins, but its underlying mechanism is poorly understood. Here, we show that Skd3 harbors both disaggregase and protein refolding activities enabled by distinct assembly states. High-resolution structures of Skd3 hexamers in distinct conformations capture ratchet-like motions that mediate substrate extraction. Unlike previously described disaggregases, Skd3 hexamers further assemble into dodecameric cages in which solubilized substrate proteins can attain near-native states. Skd3 mutants defective in dodecamer assembly retain disaggregase activity but are impaired in client refolding, linking the disaggregase and refolding activities to the hexameric and dodecameric states of Skd3, respectively. We suggest that Skd3 is a combined disaggregase and foldase, and this property is particularly suited to meet the complex proteostatic demands in the mitochondrial IMS.
Topics: Animals; Humans; Molecular Chaperones; Protein Refolding
PubMed: 37163603
DOI: 10.1126/sciadv.adf5336 -
Journal of Visualized Experiments : JoVE Dec 2017Identification of natural ligands of chemoreceptors and structural studies aimed at elucidation of the molecular basis of the ligand specificity can be greatly...
Identification of natural ligands of chemoreceptors and structural studies aimed at elucidation of the molecular basis of the ligand specificity can be greatly facilitated by the production of milligram amounts of pure, folded ligand binding domains. Attempts to heterologously express periplasmic ligand binding domains of bacterial chemoreceptors in Escherichia coli (E. coli) often result in their targeting into inclusion bodies. Here, a method is presented for protein recovery from inclusion bodies, its refolding and purification, using the periplasmic dCACHE ligand binding domain of Campylobacter jejuni (C. jejuni) chemoreceptor Tlp3 as an example. The approach involves expression of the protein of interest with a cleavable His6-tag, isolation and urea-mediated solubilisation of inclusion bodies, protein refolding by urea depletion, and purification by means of affinity chromatography, followed by tag removal and size-exclusion chromatography. The circular dichroism spectroscopy is used to confirm the folded state of the pure protein. It has been demonstrated that this protocol is generally useful for production of milligram amounts of dCACHE periplasmic ligand binding domains of other bacterial chemoreceptors in a soluble and crystallisable form.
Topics: Binding Sites; Chemoreceptor Cells; Ligands; Protein Refolding
PubMed: 29286481
DOI: 10.3791/57092 -
Methods in Enzymology 2016The diverse roles of chemokines in normal immune function and many human diseases have motivated numerous investigations into the structure and function of this family...
The diverse roles of chemokines in normal immune function and many human diseases have motivated numerous investigations into the structure and function of this family of proteins. Recombinant chemokines are often used to study how chemokines coordinate the trafficking of immune cells in various biological contexts. A reliable source of biologically active protein is vital for any in vitro or in vivo functional analysis. In this chapter, we describe a general method for the production of recombinant chemokines and robust techniques for efficient refolding that ensure consistently high biological activity. Considerations for initiating development of protocols consistent with Current Good Manufacturing Practices (cGMPs) to produce biologically active chemokines suitable for use in clinical trials are also discussed.
Topics: Chemotaxis; Chromatography, Affinity; Chromatography, High Pressure Liquid; Cyclic GMP; Disulfides; Escherichia coli; Nuclear Magnetic Resonance, Biomolecular; Protein Engineering; Protein Processing, Post-Translational; Protein Refolding; Recombinant Proteins; Reproducibility of Results
PubMed: 26921961
DOI: 10.1016/bs.mie.2015.09.031 -
Cellular and Molecular Life Sciences :... May 2006G protein-coupled receptors (GPCRS) represent a class of integral membrane proteins involved in many biological processes and pathologies. Fifty percent of all modern... (Comparative Study)
Comparative Study Review
G protein-coupled receptors (GPCRS) represent a class of integral membrane proteins involved in many biological processes and pathologies. Fifty percent of all modern drugs and almost 25% of the top 200 bestselling drugs are estimated to target GPCRs. Despite these crucial biological implications, very little is known, at atomic resolution, about the detailed molecular mechanisms by which these membrane proteins are able to recognize their extra-cellular stimuli and transmit the associated messages. Obviously, our understanding of GPCR functioning would be greatly facilitated by the availability of high-resolution three-dimensional (3D) structural data. However, expression, solubilization and purification of these membrane proteins are not easy to achieve, and at present, only one 3D structure has been determined, that of bovine rhodopsin. This review presents and compares the different successful strategies which have been applied to solubilize and purify recombinant GPCRs in the perspective of structural biology experiments.
Topics: Animals; Chromatography; Detergents; Humans; Protein Renaturation; Receptors, G-Protein-Coupled; Recombinant Proteins; Solubility
PubMed: 16568239
DOI: 10.1007/s00018-005-5557-6