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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 -
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 -
Protein Science : a Publication of the... Jul 2005A recurring obstacle for structural genomics is the expression of insoluble, aggregated proteins. In these cases, the use of alternative salvage strategies, like in...
A recurring obstacle for structural genomics is the expression of insoluble, aggregated proteins. In these cases, the use of alternative salvage strategies, like in vitro refolding, is hindered by the lack of a universal refolding method. To overcome this obstacle, fractional factorial screens have been introduced as a systematic and rapid method to identify refolding conditions. However, methodical analyses of the effectiveness of refolding reagents on large sets of proteins remain limited. In this study, we address this void by designing a fractional factorial screen to rapidly explore the effect of 14 different reagents on the refolding of 33 structurally and functionally diverse proteins. The refolding data was analyzed using statistical methods to determine the effect of each refolding additive. The screen has been miniaturized for automation resulting in reduced protein requirements and increased throughput. Our results show that the choice of pH and reducing agent had the largest impact on protein refolding. Bis-mercaptoacetamide cyclohexane (BMC) and tris (2-carboxyethylphosphine) (TCEP) were superior reductants when compared to others in the screen. BMC was particularly effective in refolding disulfide-containing proteins, while TCEP was better for nondisulfide-containing proteins. From the screen, we successfully identified a positive synergistic interaction between nondetergent sulfobetaine 201 (NDSB 201) and BMC on Cdc25A refolding. The soluble protein resulting from this interaction crystallized and yielded a 2.2 Angstroms structure. Our method, which combines a fractional factorial screen with statistical analysis of the data, provides a powerful approach for the identification of optimal refolding reagents in a general refolding screen.
Topics: Crystallography, X-Ray; Disulfides; Guanidine; Inclusion Bodies; Protein Denaturation; Protein Folding; Protein Renaturation; Proteins; Recombinant Proteins; Reducing Agents; Robotics
PubMed: 15937284
DOI: 10.1110/ps.051433205 -
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 -
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 -
Cell Stress & Chaperones Sep 2012Post-heat shock refolding of luciferase requires chaperones. Expression of a dominant negative HSF1 mutant (dnHSF1), which among other effects depletes cells of...
Post-heat shock refolding of luciferase requires chaperones. Expression of a dominant negative HSF1 mutant (dnHSF1), which among other effects depletes cells of HSF1-regulated chaperones, blocked post-heat shock refolding of luciferase targeted to the cytoplasm, nucleus, or peroxisomes, while refolding of endoplasmic reticulum (ER)-targeted luciferase was inhibited by about 50 %. Luciferase refolding in the cytoplasm could be partially restored by expression of HSPA1A and fully by both HSPA1A and DNAJB1. For full refolding of ER luciferase, HSPA1A expression sufficed. Neither nuclear nor peroxisomal refolding was rescued by HSPA1A. A stimulatory effect of DNAJB1 on post-heat shock peroxisomal luciferase refolding was seen in control cells, while refolding in the cytoplasm or nucleus in control cells was inhibited by DNAJB1 expression in the absence of added HSPA1A. HSPB1 also improved refolding of peroxisomal luciferase in control cells, but not in dnHSF1 expressing cells. HSP90, HSPA5, HSPA6, and phosphomevalonate kinase (of which the synthesis is also downregulated by dnHSF1) had no effect on peroxisomal refolding in either control or chaperone-depleted cells. The chaperone requirement for post-heat shock refolding of peroxisomal luciferase in control cells is thus unusual in that it can be augmented by DNAJB1 or HSPB1 but not by HSPA1A; in dnHSF1 expressing cells, expression of none of the (co)-chaperones tested was effective, and an as yet to be identified, HSF1-regulated function is required.
Topics: DNA-Binding Proteins; Endoplasmic Reticulum; Endoplasmic Reticulum Chaperone BiP; Genes, Reporter; HEK293 Cells; HSP40 Heat-Shock Proteins; HSP70 Heat-Shock Proteins; HSP90 Heat-Shock Proteins; Heat Shock Transcription Factors; Heat-Shock Proteins; Humans; Luciferases; Peroxisomes; Phosphotransferases (Phosphate Group Acceptor); Protein Refolding; Transcription Factors; Transfection
PubMed: 22477622
DOI: 10.1007/s12192-012-0335-5 -
Methods (San Diego, Calif.) May 2013There are many ways to present antigens to the immune system. We have used a repetitive antigen display technology that relies on the self-assembly of 60 protein chains...
There are many ways to present antigens to the immune system. We have used a repetitive antigen display technology that relies on the self-assembly of 60 protein chains into a spherical self-assembling protein nanoparticle (SAPN) to develop a vaccine against Plasmodium falciparum malaria. The protein sequence contains selected B- and T-cell epitopes of the circumsporozoite protein of P. falciparum (PfCSP) and, when assembled into a nanoparticle induces strong, long-lived and protective immune responses against the PfCSP. Here we describe the conditions needed for promoting self-assembly of a P. falciparum vaccine nanoparticle, PfCSP-KMY-SAPN, and note pitfalls that may occur when determining conditions for other SAPN vaccines. Attention was paid to selecting processes that were amenable to scale up and cGMP manufacturing.
Topics: Amino Acid Sequence; Antigens, Protozoan; Epitopes, B-Lymphocyte; Epitopes, T-Lymphocyte; Escherichia coli; Gene Expression; Humans; Malaria Vaccines; Malaria, Falciparum; Microscopy, Electron, Transmission; Molecular Sequence Data; Nanoparticles; Plasmodium falciparum; Protein Refolding; Protozoan Proteins; Vaccines, Synthetic
PubMed: 23548672
DOI: 10.1016/j.ymeth.2013.03.025 -
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 -
Biochimica Et Biophysica Acta Apr 2012Transmembrane transporters are responsible for maintaining a correct internal cellular environment. The inherent flexibility of transporters together with their... (Review)
Review
Transmembrane transporters are responsible for maintaining a correct internal cellular environment. The inherent flexibility of transporters together with their hydrophobic environment means that they are challenging to study in vitro, but recently significant progress been made. This review will focus on in vitro stability and folding studies of transmembrane alpha helical transporters, including reversible folding systems and thermal denaturation. The successful re-assembly of a small number of ATP binding cassette transporters is also described as this is a significant step forward in terms of understanding the folding and assembly of these more complex, multi-subunit proteins. The studies on transporters discussed here represent substantial advances for membrane protein studies as well as for research into protein folding. The work demonstrates that large flexible hydrophobic proteins are within reach of in vitro folding studies, thus holding promise for furthering knowledge on the structure, function and biogenesis of ubiquitous membrane transporter families. This article is part of a Special Issue entitled: Protein Folding in Membranes.
Topics: Membrane Transport Proteins; Models, Biological; Protein Denaturation; Protein Folding; Protein Refolding; Protein Stability
PubMed: 22100867
DOI: 10.1016/j.bbamem.2011.11.006 -
International Journal of Molecular... Dec 2020NanoLuc is a bioluminescent protein recently engineered for applications in molecular imaging and cellular reporter assays. Compared to other bioluminescent proteins...
NanoLuc is a bioluminescent protein recently engineered for applications in molecular imaging and cellular reporter assays. Compared to other bioluminescent proteins used for these applications, like Firefly Luciferase and Renilla Luciferase, it is ~150 times brighter, more thermally stable, and smaller. Yet, no information is known with regards to its mechanical properties, which could introduce a new set of applications for this unique protein, such as a novel biomaterial or as a substrate for protein activity/refolding assays. Here, we generated a synthetic NanoLuc derivative protein that consists of three connected NanoLuc proteins flanked by two human titin I91 domains on each side and present our mechanical studies at the single molecule level by performing Single Molecule Force Spectroscopy (SMFS) measurements. Our results show each NanoLuc repeat in the derivative behaves as a single domain protein, with a single unfolding event occurring on average when approximately 72 pN is applied to the protein. Additionally, we performed cyclic measurements, where the forces applied to a single protein were cyclically raised then lowered to allow the protein the opportunity to refold: we observed the protein was able to refold to its correct structure after mechanical denaturation only 16.9% of the time, while another 26.9% of the time there was evidence of protein misfolding to a potentially non-functional conformation. These results show that NanoLuc is a mechanically moderately weak protein that is unable to robustly refold itself correctly when stretch-denatured, which makes it an attractive model for future protein folding and misfolding studies.
Topics: Amino Acid Sequence; Base Sequence; Genetic Engineering; Humans; Luciferases; Luminescence; Luminescent Measurements; Mechanical Phenomena; Microscopy, Atomic Force; Protein Conformation; Protein Folding; Protein Refolding; Protein Stability; Protein Unfolding; Structure-Activity Relationship
PubMed: 33374567
DOI: 10.3390/ijms22010055