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Nature Communications Oct 2016Heat shock protein (Hsp)70 is a molecular chaperone that maintains protein homoeostasis during cellular stress through two opposing mechanisms: protein refolding and...
Heat shock protein (Hsp)70 is a molecular chaperone that maintains protein homoeostasis during cellular stress through two opposing mechanisms: protein refolding and degradation. However, the mechanisms by which Hsp70 balances these opposing functions under stress conditions remain unknown. Here, we demonstrate that Hsp70 preferentially facilitates protein refolding after stress, gradually switching to protein degradation via a mechanism dependent on ARD1-mediated Hsp70 acetylation. During the early stress response, Hsp70 is immediately acetylated by ARD1 at K77, and the acetylated Hsp70 binds to the co-chaperone Hop to allow protein refolding. Thereafter, Hsp70 is deacetylated and binds to the ubiquitin ligase protein CHIP to complete protein degradation during later stages. This switch is required for the maintenance of protein homoeostasis and ultimately rescues cells from stress-induced cell death in vitro and in vivo. Therefore, ARD1-mediated Hsp70 acetylation is a regulatory mechanism that temporally balances protein refolding/degradation in response to stress.
Topics: Acetylation; Animals; Apoptosis; Caspases; Cell Survival; Green Fluorescent Proteins; HEK293 Cells; HSP70 Heat-Shock Proteins; Humans; Molecular Chaperones; Mutation; N-Terminal Acetyltransferase A; N-Terminal Acetyltransferase E; Protein Binding; Protein Domains; Protein Processing, Post-Translational; Protein Refolding; RNA, Small Interfering; Stress, Physiological; Zebrafish
PubMed: 27708256
DOI: 10.1038/ncomms12882 -
BMC Structural Biology Apr 2017More than 7000 papers related to "protein refolding" have been published to date, with approximately 300 reports each year during the last decade. Whilst some of these...
BACKGROUND
More than 7000 papers related to "protein refolding" have been published to date, with approximately 300 reports each year during the last decade. Whilst some of these papers provide experimental protocols for protein refolding, a survey in the structural life science communities showed a necessity for a comprehensive database for refolding techniques. We therefore have developed a new resource - "REFOLDdb" that collects refolding techniques into a single, searchable repository to help researchers develop refolding protocols for proteins of interest.
RESULTS
We based our resource on the existing REFOLD database, which has not been updated since 2009. We redesigned the data format to be more concise, allowing consistent representations among data entries compared with the original REFOLD database. The remodeled data architecture enhances the search efficiency and improves the sustainability of the database. After an exhaustive literature search we added experimental refolding protocols from reports published 2009 to early 2017. In addition to this new data, we fully converted and integrated existing REFOLD data into our new resource. REFOLDdb contains 1877 entries as of March 17, 2017, and is freely available at http://p4d-info.nig.ac.jp/refolddb/ .
CONCLUSION
REFOLDdb is a unique database for the life sciences research community, providing annotated information for designing new refolding protocols and customizing existing methodologies. We envisage that this resource will find wide utility across broad disciplines that rely on the production of pure, active, recombinant proteins. Furthermore, the database also provides a useful overview of the recent trends and statistics in refolding technology development.
Topics: Algorithms; Databases, Protein; Humans; Internet; Protein Refolding; Proteins; User-Computer Interface
PubMed: 28438161
DOI: 10.1186/s12900-017-0074-z -
Biomolecules Feb 2014Biologically active proteins are useful for studying the biological functions of genes and for the development of therapeutic drugs and biomaterials in a biotechnology... (Review)
Review
Biologically active proteins are useful for studying the biological functions of genes and for the development of therapeutic drugs and biomaterials in a biotechnology industry. Overexpression of recombinant proteins in bacteria, such as Escherichia coli, often results in the formation of inclusion bodies, which are protein aggregates with non-native conformations. As inclusion bodies contain relatively pure and intact proteins, protein refolding is an important process to obtain active recombinant proteins from inclusion bodies. However, conventional refolding methods, such as dialysis and dilution, are time consuming and, often, recovered yields of active proteins are low, and a trial-and-error process is required to achieve success. Recently, several approaches have been reported to refold these aggregated proteins into an active form. The strategies largely aim at reducing protein aggregation during the refolding procedure. This review focuses on protein refolding techniques using chemical additives and laminar flow in microfluidic chips for the efficient recovery of active proteins from inclusion bodies.
Topics: Biochemistry; Escherichia coli; Inclusion Bodies; Protein Refolding; Recombinant Proteins
PubMed: 24970214
DOI: 10.3390/biom4010235 -
Molecules (Basel, Switzerland) May 2021The intracellular environment is overcrowded with a range of molecules (small and large), all of which influence protein conformation. As a result, understanding how...
Structural Refolding and Thermal Stability of Myoglobin in the Presence of Mixture of Crowders: Importance of Various Interactions for Protein Stabilization in Crowded Conditions.
The intracellular environment is overcrowded with a range of molecules (small and large), all of which influence protein conformation. As a result, understanding how proteins fold and stay functional in such crowded conditions is essential. Several in vitro experiments have looked into the effects of macromolecular crowding on different proteins. However, there are hardly any reports regarding small molecular crowders used alone and in mixtures to observe their effects on the structure and stability of the proteins, which mimics of the cellular conditions. Here we investigate the effect of different mixtures of crowders, ethylene glycol (EG) and its polymer polyethylene glycol (PEG 400 Da) on the structural and thermal stability of myoglobin (Mb). Our results show that monomer (EG) has no significant effect on the structure of Mb, while the polymer disrupts its structure and decreases its stability. Conversely, the additive effect of crowders showed structural refolding of the protein to some extent. Moreover, the calorimetric binding studies of the protein showed very weak interactions with the mixture of crowders. Usually, we can assume that soft interactions induce structural perturbations while exclusion volume effects stabilize the protein structure; therefore, we hypothesize that under in vivo crowded conditions, both phenomena occur and maintain the stability and function of proteins.
Topics: Animals; Dynamic Light Scattering; Ethylene Glycol; Fluorescence; Guanidine; Horses; Hydrodynamics; Macromolecular Substances; Molecular Docking Simulation; Myoglobin; Polyethylene Glycols; Protein Conformation; Protein Denaturation; Protein Refolding; Protein Stability; Temperature
PubMed: 34068693
DOI: 10.3390/molecules26092807 -
PloS One 2018RIP2, one of the RIP kinases, interacts with p75 neurotrophin receptor, regulating the neuron survival, and with NOD1 and NOD2 proteins, causing the innate immune...
RIP2, one of the RIP kinases, interacts with p75 neurotrophin receptor, regulating the neuron survival, and with NOD1 and NOD2 proteins, causing the innate immune response against gram-negative and gram-positive bacteria via its caspase recruitment domain (CARD). This makes RIP2 a prospective target for novel therapies, aimed to modulate the inflammatory diseases and neurogenesis/neurodegeneration. Several studies report the problems with the stability of human RIP2 CARD and its production in bacterial hosts, which is a prerequisite for the structural investigation with solution NMR spectroscopy. In the present work, we report the high yield production and refolding protocols and resolve the structure of rat RIP2 CARD. The structure reveals the important differences to the previously published conformation of the homologous human protein. Using solution NMR, we characterized the intramolecular mobility and pH-dependent behavior of RIP2 CARD, and found the propensity of the protein to form high-order oligomers at physiological pH while being monomeric under acidic conditions. The oligomerization of protein may be explained, based on the electrostatic properties of its surface. Analysis of the structure and sequences of homologous proteins reveals the residues which are significant for the unusual fold of RIP2 CARD domains from different species. The high-throughput protein production/refolding protocols and proposed explanation for the protein oligomerization, provide an opportunity to design the stabilized variants of RIP2 CARD, which could be used to study the structural details of RIP2/NOD1/NOD2 interaction and perform the rational drug design.
Topics: Amino Acid Sequence; Caspase Activation and Recruitment Domain; Hydrogen-Ion Concentration; Magnetic Resonance Spectroscopy; Models, Molecular; Nod1 Signaling Adaptor Protein; Nod2 Signaling Adaptor Protein; Protein Binding; Protein Multimerization; Protein Refolding; Receptor-Interacting Protein Serine-Threonine Kinase 2; Sequence Homology, Amino Acid; Solutions; Static Electricity
PubMed: 30352081
DOI: 10.1371/journal.pone.0206244 -
Scientific Reports Aug 2017Refolding of proteins derived from inclusion bodies is very promising as it can provide a reliable source of target proteins of high purity. However, inclusion...
Refolding of proteins derived from inclusion bodies is very promising as it can provide a reliable source of target proteins of high purity. However, inclusion body-based protein production is often limited by the lack of techniques for the detection of correctly refolded protein. Thus, the selection of the refolding conditions is mostly achieved using trial and error approaches and is thus a time-consuming process. In this study, we use the latest developments in the differential scanning fluorimetry guided refolding approach as an analytical method to detect correctly refolded protein. We describe a systematic buffer screen that contains a 96-well primary pH-refolding screen in conjunction with a secondary additive screen. Our research demonstrates that this approach could be applied for determining refolding conditions for several proteins. In addition, it revealed which "helper" molecules, such as arginine and additives are essential. Four different proteins: HA-RBD, MDM2, IL-17A and PD-L1 were used to validate our refolding approach. Our systematic protocol evaluates the impact of the "helper" molecules, the pH, buffer system and time on the protein refolding process in a high-throughput fashion. Finally, we demonstrate that refolding time and a secondary thermal shift assay buffer screen are critical factors for improving refolding efficiency.
Topics: Buffers; Chromatography, Gel; Hydrogen-Ion Concentration; Models, Molecular; Protein Conformation; Protein Denaturation; Protein Interaction Domains and Motifs; Protein Refolding; Proteins; Recombinant Proteins; Solubility
PubMed: 28839267
DOI: 10.1038/s41598-017-09687-z -
PloS One 2011Nerve Growth Factor is an essential protein that supports neuronal survival during development and influences neuronal function throughout adulthood, both in the central...
Nerve Growth Factor is an essential protein that supports neuronal survival during development and influences neuronal function throughout adulthood, both in the central and peripheral nervous system. The unprocessed precursor of NGF, proNGF, seems to be endowed with biological functions distinct from those of the mature protein, such as chaperone-like activities and apoptotic and/or neurotrophic properties. We have previously suggested, based on Small Angle X-ray Scattering data, that recombinant murine proNGF has features typical of an intrinsically unfolded protein. Using complementary biophysical techniques, we show here new evidence that clarifies and widens this hypothesis through a detailed comparison of the structural properties of NGF and proNGF. Our data provide direct information about the dynamic properties of the pro-peptide and indicate that proNGF assumes in solution a compact globular conformation. The N-terminal pro-peptide extension influences the chemical environment of the mature protein and protects the protein from proteolytic digestion. Accordingly, we observe that unfolding of proNGF involves a two-steps mechanism. The distinct structural properties of proNGF as compared to NGF agree with and rationalise a different functional role of the precursor.
Topics: Animals; Calorimetry, Differential Scanning; Circular Dichroism; Culture Media; Diffusion; Magnetic Resonance Spectroscopy; Mice; Models, Molecular; Molecular Weight; Nerve Growth Factor; Peptides; Protein Precursors; Protein Refolding; Protein Stability; Protein Structure, Secondary; Protein Structure, Tertiary; Protein Unfolding; Proteolysis; Solutions; Spectroscopy, Fourier Transform Infrared; Trypsin
PubMed: 21818348
DOI: 10.1371/journal.pone.0022615 -
International Review of Cell and... 2013This review focuses on the current view of the interaction between the β-barrel scaffold of fluorescent proteins and their unique chromophore located in the internal... (Review)
Review
This review focuses on the current view of the interaction between the β-barrel scaffold of fluorescent proteins and their unique chromophore located in the internal helix. The chromophore originates from the polypeptide chain and its properties are influenced by the surrounding protein matrix of the β-barrel. On the other hand, it appears that a chromophore tightens the β-barrel scaffold and plays a crucial role in its stability. Furthermore, the presence of a mature chromophore causes hysteresis of protein unfolding and refolding. We survey studies measuring protein unfolding and refolding using traditional methods as well as new approaches, such as mechanical unfolding and reassembly of truncated fluorescent proteins. We also analyze models of fluorescent protein unfolding and refolding obtained through different approaches, and compare the results of protein folding in vitro to co-translational folding of a newly synthesized polypeptide chain.
Topics: Luminescent Proteins; Models, Molecular; Protein Refolding; Protein Stability; Protein Structure, Secondary; Protein Unfolding
PubMed: 23351712
DOI: 10.1016/B978-0-12-407699-0.00004-2 -
PloS One 2019In this study we evaluated the association of high hydrostatic pressure (HHP) and alkaline pH as a minimally denaturing condition for the solubilization of inclusion...
In this study we evaluated the association of high hydrostatic pressure (HHP) and alkaline pH as a minimally denaturing condition for the solubilization of inclusion bodies (IBs) generated by recombinant proteins expressed by Escherichia coli strains. The method was successfully applied to a recombinant form of the dengue virus (DENV) non-structural protein 1 (NS1). The minimal pH for IBs solubilization at 1 bar was 12 while a pH of 10 was sufficient for solubilization at HHP: 2.4 kbar for 90 min and 0.4 kbar for 14 h 30 min. An optimal refolding condition was achieved by compression of IBs at HHP and pH 10.5 in the presence of arginine, oxidized and reduced glutathiones, providing much higher yields (up to 8-fold) than association of HHP and GdnHCl via an established protocol. The refolded NS1, 109 ± 9.5 mg/L bacterial culture was recovered mainly as monomer and dimer, corresponding up to 90% of the total protein and remaining immunologically active. The proposed conditions represent an alternative for the refolding of immunologically active recombinant proteins expressed as IBs.
Topics: Dengue Virus; Hydrogen-Ion Concentration; Hydrostatic Pressure; Protein Refolding; Recombinant Proteins; Viral Nonstructural Proteins
PubMed: 30682103
DOI: 10.1371/journal.pone.0211162 -
Nature Communications Jul 2016Talin, a force-bearing cytoplasmic adapter essential for integrin-mediated cell adhesion, links the actin cytoskeleton to integrin-based cell-extracellular matrix...
Talin, a force-bearing cytoplasmic adapter essential for integrin-mediated cell adhesion, links the actin cytoskeleton to integrin-based cell-extracellular matrix adhesions at the plasma membrane. Its C-terminal rod domain, which contains 13 helical bundles, plays important roles in mechanosensing during cell adhesion and spreading. However, how the structural stability and transition kinetics of the 13 helical bundles of talin are utilized in the diverse talin-dependent mechanosensing processes remains poorly understood. Here we report the force-dependent unfolding and refolding kinetics of all talin rod domains. Using experimentally determined kinetics parameters, we determined the dynamics of force fluctuation during stretching of talin under physiologically relevant pulling speeds and experimentally measured extension fluctuation trajectories. Our results reveal that force-dependent stochastic unfolding and refolding of talin rod domains make talin a very effective force buffer that sets a physiological force range of only a few pNs in the talin-mediated force transmission pathway.
Topics: Animals; Binding Sites; Biomechanical Phenomena; Cloning, Molecular; Endopeptidases; Escherichia coli; Gene Expression; Glutathione Transferase; Kinetics; Mice; Models, Molecular; Protein Binding; Protein Folding; Protein Interaction Domains and Motifs; Protein Refolding; Protein Structure, Secondary; Recombinant Fusion Proteins; Single Molecule Imaging; Stress, Mechanical; Talin; Thermodynamics
PubMed: 27384267
DOI: 10.1038/ncomms11966