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Proceedings of the National Academy of... Nov 2022The journey by which proteins navigate their energy landscapes to their native structures is complex, involving (and sometimes requiring) many cellular factors and...
The journey by which proteins navigate their energy landscapes to their native structures is complex, involving (and sometimes requiring) many cellular factors and processes operating in partnership with a given polypeptide chain's intrinsic energy landscape. The cytosolic environment and its complement of chaperones play critical roles in granting many proteins safe passage to their native states; however, it is challenging to interrogate the folding process for large numbers of proteins in a complex background with most biophysical techniques. Hence, most chaperone-assisted protein refolding studies are conducted in defined buffers on single purified clients. Here, we develop a limited proteolysis-mass spectrometry approach paired with an isotope-labeling strategy to globally monitor the structures of refolding proteins in the cytosolic medium and with the chaperones, GroEL/ES (Hsp60) and DnaK/DnaJ/GrpE (Hsp70/40). GroEL can refold the majority (85%) of the proteins for which we have data and is particularly important for restoring acidic proteins and proteins with high molecular weight, trends that come to light because our assay measures the structural outcome of the refolding process itself, rather than binding or aggregation. For the most part, DnaK and GroEL refold a similar set of proteins, supporting the view that despite their vastly different structures, these two chaperones unfold misfolded states, as one mechanism in common. Finally, we identify a cohort of proteins that are intransigent to being refolded with either chaperone. We suggest that these proteins may fold most efficiently cotranslationally, and then remain kinetically trapped in their native conformations.
Topics: Cytosol; Escherichia coli; Escherichia coli Proteins; Heat-Shock Proteins; Molecular Chaperones; Protein Refolding; Proteome
PubMed: 36417429
DOI: 10.1073/pnas.2210536119 -
Cell Stress & Chaperones Jan 2019Hsp70 chaperone systems are very versatile machines present in nearly all living organisms and in nearly all intracellular compartments. They function in many...
Hsp70 chaperone systems are very versatile machines present in nearly all living organisms and in nearly all intracellular compartments. They function in many fundamental processes through their facilitation of protein (re)folding, trafficking, remodeling, disaggregation, and degradation. Hsp70 machines are regulated by co-chaperones. J-domain containing proteins (JDPs) are the largest family of Hsp70 co-chaperones and play a determining role functionally specifying and directing Hsp70 functions. Many features of JDPs are not understood; however, a number of JDP experts gathered at a recent CSSI-sponsored workshop in Gdansk (Poland) to discuss various aspects of J-domain protein function, evolution, and structure. In this report, we present the main findings and the consensus reached to help direct future developments in the field of Hsp70 research.
Topics: Animals; Disease; Evolution, Molecular; HSP70 Heat-Shock Proteins; Humans; Protein Aggregates; Protein Domains; Protein Refolding
PubMed: 30478692
DOI: 10.1007/s12192-018-0948-4 -
Revista Argentina de Microbiologia 2016Bacillus anthracis protective antigen (PA) is a well known and relevant immunogenic protein that is the basis for both anthrax vaccines and diagnostic methods. Properly...
Bacillus anthracis protective antigen (PA) is a well known and relevant immunogenic protein that is the basis for both anthrax vaccines and diagnostic methods. Properly folded antigenic PA is necessary for these applications. In this study a high level of PA was obtained in recombinant Escherichia coli. The protein was initially accumulated in inclusion bodies, which facilitated its efficient purification by simple washing steps; however, it could not be recognized by specific antibodies. Refolding conditions were subsequently analyzed in a high-throughput manner that enabled nearly a hundred different conditions to be tested simultaneously. The recovery of the ability of PA to be recognized by antibodies was screened by dot blot using a coefficient that provided a measure of properly refolded protein levels with a high degree of discrimination. The best refolding conditions resulted in a tenfold increase in the intensity of the dot blot compared to the control. The only refolding additive that consistently yielded good results was L-arginine. The statistical analysis identified both cooperative and negative interactions between the different refolding additives. The high-throughput approach described in this study that enabled overproduction, purification and refolding of PA in a simple and straightforward manner, can be potentially useful for the rapid screening of adequate refolding conditions for other overexpressed antigenic proteins.
Topics: Antigens, Bacterial; Bacillus anthracis; Models, Molecular; Protein Refolding
PubMed: 26777581
DOI: 10.1016/j.ram.2015.10.004 -
Postepy Higieny I Medycyny... Jun 2012Recombinant proteins and enzymes are commonly used in many areas of our life, such as diagnostics, industry and medicine, due to heterologous synthesis in prokaryotic... (Review)
Review
Recombinant proteins and enzymes are commonly used in many areas of our life, such as diagnostics, industry and medicine, due to heterologous synthesis in prokaryotic expression systems. However, a high expression level of foreign protein in bacteria cells results in formation of inactive and insoluble aggregates--inclusion bodies. Reactivation of aggregated proteins is a complex and time-consuming process. Every protein requires experimental optimization of the process conditions. The choice of the refolding method depends on the type of recombinant protein and its physical, chemical and biological properties. Recovery of the activity of proteins accumulated in inclusion bodies can be divided into 4 steps: 1) inclusion bodies isolation, 2) solubilization of aggregates, 3) renaturation, 4) purification of catalytically active molecules. Efficiency of the refolding process depends on many physical factors and chemical and biological agents. The above parameters determine the time of the folding and prevent protein aggregation. They also assist the folding and have an influence on the solubility and stability of native molecules. To date, dilution, dialysis and chromatography are the most often used methods for protein refolding.
Topics: Chromatography; Dialysis; Escherichia coli; Inclusion Bodies; Molecular Chaperones; Protein Renaturation; Recombinant Proteins; Solubility
PubMed: 22706118
DOI: 10.5604/17322693.999918 -
BMC Biotechnology Dec 2018Proteins in inclusion bodies (IBs) present native-like secondary structures. However, chaotropic agents at denaturing concentrations, which are widely used for IB...
BACKGROUND
Proteins in inclusion bodies (IBs) present native-like secondary structures. However, chaotropic agents at denaturing concentrations, which are widely used for IB solubilization and subsequent refolding, unfold these secondary structures. Removal of the chaotropes frequently causes reaggregation and poor recovery of bioactive proteins. High hydrostatic pressure (HHP) and alkaline pH are two conditions that, in the presence of low level of chaotropes, have been described as non-denaturing solubilization agents. In the present study we evaluated the strategy of combination of HHP and alkaline pH on the solubilization of IB using as a model an antigenic form of the zika virus (ZIKV) non-structural 1 (NS1) protein.
RESULTS
Pressure-treatment (2.4 kbar) of NS1-IBs at a pH of 11.0 induced a low degree of NS1 unfolding and led to solubilization of the IBs, mainly into monomers. After dialysis at pH 8.5, NS1 was refolded and formed soluble oligomers. High (up to 68 mg/liter) NS1 concentrations were obtained by solubilization of NS1-IBs at pH 11 in the presence of arginine (Arg) with a final yield of approximately 80% of total protein content. The process proved to be efficient, quick and did not require further purification steps. Refolded NS1 preserved biological features regarding reactivity with antigen-specific antibodies, including sera of ZIKV-infected patients. The method resulted in an increase of approximately 30-fold over conventional IB solubilization-refolding methods.
CONCLUSIONS
The present results represent an innovative non-denaturing protein refolding process by means of the concomitant use of HHP and alkaline pH. Application of the reported method allowed the recovery of ZIKV NS1 at a condition that maintained the antigenic properties of the protein.
Topics: Alkalies; Biochemistry; Hydrostatic Pressure; Inclusion Bodies; Protein Refolding; Protein Structure, Secondary; Solubility; Viral Nonstructural Proteins; Zika Virus
PubMed: 30541520
DOI: 10.1186/s12896-018-0486-2 -
Nature Communications Oct 2019Maintenance of cellular proteostasis is achieved by a multi-layered quality control network, which counteracts the accumulation of misfolded proteins by refolding and...
Maintenance of cellular proteostasis is achieved by a multi-layered quality control network, which counteracts the accumulation of misfolded proteins by refolding and degradation pathways. The organized sequestration of misfolded proteins, actively promoted by cellular sequestrases, represents a third strategy of quality control. Here we determine the role of sequestration within the proteostasis network in Saccharomyces cerevisiae and the mechanism by which it occurs. The Hsp42 and Btn2 sequestrases are functionally intertwined with the refolding activity of the Hsp70 system. Sequestration of misfolded proteins by Hsp42 and Btn2 prevents proteostasis collapse and viability loss in cells with limited Hsp70 capacity, likely by shielding Hsp70 from misfolded protein overload. Btn2 has chaperone and sequestrase activity and shares features with small heat shock proteins. During stress recovery Btn2 recruits the Hsp70-Hsp104 disaggregase by directly interacting with the Hsp70 co-chaperone Sis1, thereby shunting sequestered proteins to the refolding pathway.
Topics: Amino Acid Transport Systems; HSP40 Heat-Shock Proteins; HSP70 Heat-Shock Proteins; Heat-Shock Proteins; Protein Refolding; Proteostasis; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins
PubMed: 31649258
DOI: 10.1038/s41467-019-12868-1 -
Acta Crystallographica. Section F,... Mar 2014In Gram-negative bacteria, the assembly of outer membrane proteins (OMPs) requires a five-protein β-barrel assembly machinery (BAM) complex, of which BamA is an...
Refolding, crystallization and preliminary X-ray crystallographic studies of the β-barrel domain of BamA, a membrane protein essential for outer membrane protein biogenesis.
In Gram-negative bacteria, the assembly of outer membrane proteins (OMPs) requires a five-protein β-barrel assembly machinery (BAM) complex, of which BamA is an essential and evolutionarily conserved integral outer membrane protein. Here, the refolding, crystallization and preliminary X-ray crystallographic characterization of the β-barrel domain of BamA from Escherichia coli (EcBamA) are reported. Native and selenomethionine-substituted EcBamA proteins were crystallized at 16°C and X-ray diffraction data were collected to 2.6 and 3.7 Å resolution, respectively. The native crystals belonged to space group P21212, with unit-cell parameters a = 118.492, b = 159.883, c = 56.000 Å and two molecules in one asymmetric unit; selenomethionine-substituted protein crystals belonged to space group P4322, with unit-cell parameters a = b = 163.162, c = 46.388 Å and one molecule in one asymmetric unit. Initial phases for EcBamA β-barrel domain were obtained from a SeMet SAD data set. These preliminary X-ray crystallographic studies paved the way for further structural determination of the β-barrel domain of EcBamA.
Topics: Amino Acid Sequence; Bacterial Outer Membrane Proteins; Chromatography, Gel; Conserved Sequence; Crystallization; Crystallography, X-Ray; Escherichia coli; Escherichia coli Proteins; Molecular Sequence Data; Protein Biosynthesis; Protein Refolding; Protein Structure, Secondary
PubMed: 24598928
DOI: 10.1107/S2053230X14003008 -
Biophysical Journal Apr 2017The strong and usually denaturing interaction between anionic surfactants (AS) and proteins/enzymes has both benefits and drawbacks: for example, it is put to good use...
The strong and usually denaturing interaction between anionic surfactants (AS) and proteins/enzymes has both benefits and drawbacks: for example, it is put to good use in electrophoretic mass determinations but limits enzyme efficiency in detergent formulations. Therefore, studies of the interactions between proteins and AS as well as nonionic surfactants (NIS) are of both basic and applied relevance. The AS sodium dodecyl sulfate (SDS) denatures and unfolds globular proteins under most conditions. In contrast, NIS such as octaethylene glycol monododecyl ether (CE) and dodecyl maltoside (DDM) protect bovine serum albumin (BSA) from unfolding in SDS. Membrane proteins denatured in SDS can also be refolded by addition of NIS. Here, we investigate whether globular proteins unfolded by SDS can be refolded upon addition of CE and DDM. Four proteins, BSA, α-lactalbumin (αLA), lysozyme, and β-lactoglobulin (βLG), were studied by small-angle x-ray scattering and both near- and far-UV circular dichroism. All proteins and their complexes with SDS were attempted to be refolded by the addition of CE, while DDM was additionally added to SDS-denatured αLA and βLG. Except for αLA, the proteins did not interact with NIS alone. For all proteins, the addition of NIS to the protein-SDS samples resulted in extraction of the SDS from the protein-SDS complexes and refolding of βLG, BSA, and lysozyme, while αLA changed to its NIS-bound state instead of the native state. We conclude that NIS competes with globular proteins for association with SDS, making it possible to release and refold SDS-denatured proteins by adding sufficient amounts of NIS, unless the protein also interacts with NIS alone.
Topics: Animals; Cattle; Chickens; Circular Dichroism; Egg Proteins; Ethylene Glycols; Glucosides; Lactalbumin; Lactoglobulins; Micelles; Milk Proteins; Muramidase; Protein Refolding; Protein Unfolding; Scattering, Small Angle; Serum Albumin; Sodium Dodecyl Sulfate; Surface-Active Agents; X-Ray Diffraction
PubMed: 28445752
DOI: 10.1016/j.bpj.2017.03.013 -
Proceedings of the National Academy of... Aug 2021DegP is an oligomeric protein with dual protease and chaperone activity that regulates protein homeostasis and virulence factor trafficking in the periplasm of...
DegP is an oligomeric protein with dual protease and chaperone activity that regulates protein homeostasis and virulence factor trafficking in the periplasm of gram-negative bacteria. A number of oligomeric architectures adopted by DegP are thought to facilitate its function. For example, DegP can form a "resting" hexamer when not engaged to substrates, mitigating undesired proteolysis of cellular proteins. When bound to substrate proteins or lipid membranes, DegP has been shown to populate a variety of cage- or bowl-like oligomeric states that have increased proteolytic activity. Though a number of DegP's substrate-engaged structures have been robustly characterized, detailed mechanistic information underpinning its remarkable oligomeric plasticity and the corresponding interplay between these dynamics and biological function has remained elusive. Here, we have used a combination of hydrodynamics and NMR spectroscopy methodologies in combination with cryogenic electron microscopy to shed light on the apo-DegP self-assembly mechanism. We find that, in the absence of bound substrates, DegP populates an ensemble of oligomeric states, mediated by self-assembly of trimers, that are distinct from those observed in the presence of substrate. The oligomeric distribution is sensitive to solution ionic strength and temperature and is shifted toward larger oligomeric assemblies under physiological conditions. Substrate proteins may guide DegP toward canonical cage-like structures by binding to these preorganized oligomers, leading to changes in conformation. The properties of DegP self-assembly identified here suggest that apo-DegP can rapidly shift its oligomeric distribution in order to respond to a variety of biological insults.
Topics: Cryoelectron Microscopy; Dynamic Light Scattering; Heat-Shock Proteins; Molecular Chaperones; Mutation; Nuclear Magnetic Resonance, Biomolecular; Osmolar Concentration; Periplasmic Proteins; Protein Domains; Protein Refolding; Serine Endopeptidases; Temperature
PubMed: 34362850
DOI: 10.1073/pnas.2109732118 -
Protein Expression and Purification Apr 2012Xylella fastidiosa is a Gram-negative xylem-limited plant pathogenic bacterium responsible for several economically important crop diseases. Here, we present a novel and...
A novel protein refolding protocol for the solubilization and purification of recombinant peptidoglycan-associated lipoprotein from Xylella fastidiosa overexpressed in Escherichia coli.
Xylella fastidiosa is a Gram-negative xylem-limited plant pathogenic bacterium responsible for several economically important crop diseases. Here, we present a novel and efficient protein refolding protocol for the solubilization and purification of recombinant X. fastidiosa peptidoglycan-associated lipoprotein (XfPal). Pal is an outer membrane protein that plays important roles in maintaining the integrity of the cell envelope and in bacterial pathogenicity. Because Pal has a highly hydrophobic N-terminal domain, the heterologous expression studies necessary for structural and functional protein characterization are laborious once the recombinant protein is present in inclusion bodies. Our protocol based on the denaturation of the XfPal-enriched inclusion bodies with 8M urea followed by buffer-exchange steps via dialysis proved effective for the solubilization and subsequent purification of XfPal, allowing us to obtain a large amount of relatively pure and folded protein. In addition, XfPal was biochemically and functionally characterized. The method for purification reported herein is valuable for further research on the three-dimensional structure and function of Pal and other outer membrane proteins and can contribute to a better understanding of the role of these proteins in bacterial pathogenicity, especially with regard to the plant pathogen X. fastidiosa.
Topics: Amino Acid Sequence; Bacterial Proteins; Chromatography, Gel; Escherichia coli; Lipoproteins; Molecular Sequence Data; Peptidoglycan; Protein Binding; Protein Refolding; Protein Structure, Quaternary; Protein Structure, Secondary; Sequence Homology, Amino Acid; Solubility; Xylella
PubMed: 22306742
DOI: 10.1016/j.pep.2012.01.010