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PloS One 2021Aiming at streamlining GPCR production from E. coli inclusion bodies for structural analysis, we present a generic approach to assess and optimize refolding yield...
Aiming at streamlining GPCR production from E. coli inclusion bodies for structural analysis, we present a generic approach to assess and optimize refolding yield through thermostability analysis. Since commonly used hydrophobic dyes cannot be applied as probes for membrane protein unfolding, we adapted a technique based on reacting cysteins exposed upon thermal denaturation with fluorescent 7-Diethylamino-3-(4-maleimidophenyl)-4-methylcoumarin (CPM). Successful expression, purification and refolding is shown for two G protein-coupled receptors (GPCR), the sphingosine-1-phosphate receptor S1P1, and the orphan receptor GPR3. Refolded receptors were subjected to lipidic cubic phase crystallization screening.
Topics: Escherichia coli; Escherichia coli Proteins; Inclusion Bodies; Protein Refolding; Receptors, G-Protein-Coupled; Sphingosine-1-Phosphate Receptors
PubMed: 33626080
DOI: 10.1371/journal.pone.0247689 -
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 -
Methods in Molecular Biology (Clifton,... 2022Aspartic proteases (APs) are widely distributed in plants. The large majority of genes encoding putative APs exhibit distinct features when compared with the so-called...
Aspartic proteases (APs) are widely distributed in plants. The large majority of genes encoding putative APs exhibit distinct features when compared with the so-called typical APs, and have been grouped as atypical and nucellin-like APs. Remarkably, a diverse pattern of enzymatic properties, subcellular localizations, and biological functions are emerging for these proteases, illustrating the functional complexity among plant pepsin-like proteases. However, many key questions regarding the structure-function relationships of plant APs remain unanswered. Therefore, the expression of these enzymes in heterologous systems is a valuable strategy to unfold the unique features/biochemical properties among members of this family of proteases. Here, we describe our protocol for the production and purification of recombinant plant APs, using a procedure where the protein is refolded from inclusion bodies by dialysis. This method allows the production of untagged versions of the target protease, which has revealed to be critical to disclose differences in processing/activation requirements between plant APs. The protocol includes protein expression, washing and solubilization of inclusion bodies, refolding by dialysis, and a protein purification method. Specific considerations on critical aspects of the refolding process and further suggestions for evaluation of the final recombinant product are also provided.
Topics: Aspartic Acid Proteases; Escherichia coli; Inclusion Bodies; Plants; Protein Refolding; Recombinant Proteins; Renal Dialysis
PubMed: 35583770
DOI: 10.1007/978-1-0716-2079-3_3 -
Biological Chemistry Mar 2019The minor capsid protein L2 of papillomaviruses exhibits multiple functions during viral entry including membrane interaction. Information on the protein is scarce,...
The minor capsid protein L2 of papillomaviruses exhibits multiple functions during viral entry including membrane interaction. Information on the protein is scarce, because of its high tendency of aggregation. We determined suitable conditions to produce a functional human papillomavirus (HPV) 16 L2 protein and thereby provide the opportunity for extensive in vitro analysis with respect to structural and biochemical information on L2 proteins and mechanistic details in viral entry. We produced the L2 protein of high-risk HPV 16 in Escherichia coli as inclusion bodies and purified the protein under denaturing conditions. A successive buffer screen resulted in suitable conditions for the biophysical characterization of 16L2. Analytical ultracentrifugation of the refolded protein showed a homogenous monomeric species. Furthermore, refolded 16L2 shows secondary structure elements. The N-terminal region including the proposed transmembrane region of 16L2 shows alpha-helical characteristics. However, overall 16L2 appears largely unstructured. Refolded 16L2 is capable of binding to DNA indicating that the putative DNA-binding regions are accessible in refolded 16L2. Further the refolded protein interacts with liposomal membranes presumably via the proposed transmembrane region at neutral pH without structural changes. This indicates that 16L2 can initially interact with membranes via pre-existing structural features.
Topics: Capsid Proteins; DNA, Viral; Humans; Liposomes; Nucleic Acids; Oncogene Proteins, Viral; Protein Refolding; Protein Stability; Protein Structure, Secondary
PubMed: 30375341
DOI: 10.1515/hsz-2018-0311 -
ACS Nano Oct 2017The folding process of a protein is inherently error-prone, owing to the large number of possible conformations that a protein chain can adopt. Partially folded or...
The folding process of a protein is inherently error-prone, owing to the large number of possible conformations that a protein chain can adopt. Partially folded or misfolded proteins typically expose hydrophobic surfaces and tend to form dysfunctional protein aggregates. Therefore, materials that can stabilize unfolded proteins and then efficiently assist them refolding to its bioactive form are of significant interest. Inspired by natural chaperonins, we have synthesized a series of polymeric nanochaperones that can facilitate the refolding of denatured proteins with a high recovery efficiency (up to 97%). Such nanochaperones possess phase-separated structure with hydrophobic microdomains on the surface. This structure allows nanochaperones to stabilize denatured proteins by binding them to the hydrophobic microdomains. We have also investigated the mechanism by which nanochaperones assist the protein refolding and established the design principles of nanochaperones in order to achieve effective recovery of a certain protein from their denatured forms. With a carefully designed composition of the microdomains according to the surface properties of the client proteins, the binding affinity between the hydrophobic microdomain and the denatured protein molecules can be tuned precisely, which enables the self-sorting of the polypeptides and the refolding of the proteins into their bioactive states. This work provides a feasible and effective strategy to recover inclusion bodies to their bioactive forms, which has potential to reduce the cost of the manufacture of recombinant proteins significantly.
Topics: Molecular Chaperones; Muramidase; Nanoparticles; Particle Size; Protein Denaturation; Protein Refolding; Surface Properties; Temperature
PubMed: 28968070
DOI: 10.1021/acsnano.7b05947 -
Methods in Enzymology 2015OmpG is a pore-forming protein from E. coli outer membranes. Unlike the classical outer membrane porins, which are trimers, the OmpG channel is a monomeric β-barrel...
OmpG is a pore-forming protein from E. coli outer membranes. Unlike the classical outer membrane porins, which are trimers, the OmpG channel is a monomeric β-barrel made of 14 antiparallel β-strands with short periplasmic turns and longer extracellular loops. The channel activity of OmpG is pH dependent and the channel is gated by the extracellular loop L6. At neutral/high pH, the channel is open and permeable for substrate molecules with a size up to 900 Da. At acidic pH, loop L6 folds across the channel and blocks the pore. The channel blockage at acidic pH appears to be triggered by the protonation of a histidine pair on neighboring β-strands, which repel one another, resulting in the rearrangement of loop L6 and channel closure. OmpG was purified by refolding from inclusion bodies and crystallized in two and three dimensions. Crystallization and analysis by electron microscopy and X-ray crystallography revealed the fundamental mechanisms essential for the channel activity.
Topics: Bacterial Outer Membrane Proteins; Chromatography, Gel; Chromatography, Ion Exchange; Circular Dichroism; Cryoelectron Microscopy; Crystallization; Crystallography, X-Ray; Electrophoresis; Escherichia coli; Escherichia coli Proteins; Models, Molecular; Porins; Protein Refolding; Protein Structure, Secondary
PubMed: 25950964
DOI: 10.1016/bs.mie.2015.01.018 -
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 -
The FEBS Journal May 2017The ubiquitous eukaryotic 14-3-3 proteins coordinate multiple cellular processes due to their well-known regulatory function, which is based on specific recognition of... (Comparative Study)
Comparative Study Review
The ubiquitous eukaryotic 14-3-3 proteins coordinate multiple cellular processes due to their well-known regulatory function, which is based on specific recognition of phosphorylated motifs in their partners. In this context, 14-3-3 proteins have been called 'chaperones'. Although in the classical meaning this is not fully correct, recent studies have revealed that they can indeed be an integral part of the protein quality control system, as they (a) display ATP-independent anti-aggregation ('holdase') activity, similar to that of the unrelated small heat shock proteins, (b) assist in clearing misfolded proteins by directing them to proteasomes or aggresomes, (c) cooperate with classical chaperones for substrate refolding, and also (d) are associated with neurodegenerative disorders by affecting aggregation of tau, prion protein, α-synuclein, huntingtin, etc. Importantly, these activities are usually independent of substrate phosphorylation and therefore should be considered as distinct, 'moonlighting' functions of 14-3-3 proteins that mimic and complement the functions of dedicated molecular chaperones. Although the precise mechanism of this activity is still unknown, it has been shown that it is not dependent on the unstructured C-terminal region or the amphipathic phosphopeptide-binding groove. However, since disassembly of 14-3-3 dimers significantly increases their chaperone-like activity, the dimer interface, located in the N terminus, possessing a high disorder propensity and pronounced hydrophobicity, is likely to be involved. Various factors affecting the oligomeric status of 14-3-3 proteins can thus regulate the balance between regulatory phosphomotif binding and genuine chaperone-like activity. Understanding the latter mode of 14-3-3 functioning is fundamental to defining the underlying molecular mechanisms for a range of human disorders.
Topics: 14-3-3 Proteins; Amino Acid Motifs; Animals; Binding Sites; Dimerization; Humans; Hydrophobic and Hydrophilic Interactions; Models, Molecular; Molecular Chaperones; Phosphorylation; Protein Conformation; Protein Interaction Domains and Motifs; Protein Multimerization; Protein Processing, Post-Translational; Protein Refolding
PubMed: 27973707
DOI: 10.1111/febs.13986 -
International Journal of Biological... Dec 2021Formation of protein aggregates as inclusion bodies (IBs) still poses a major hurdle in the recovery of bioactive proteins from E. coli. Despite the development of many...
Formation of protein aggregates as inclusion bodies (IBs) still poses a major hurdle in the recovery of bioactive proteins from E. coli. Despite the development of many mild solubilization buffers in last two decades, high-throughput recovery of functional protein from wide range of IBs is still a challenge at an academic and industrial scale. Herein, a novel formulation for improved recovery of bioactive protein from variety of bacterial IBs is developed. This novel formulation is comprised of 20% trifluoroethanol, 20% n-propanol and 2 M urea at pH 12.5 which disrupts the major dominant forces involved in protein aggregation. An extensive comparative study of novel formulation conducted on different IBs demonstrates its high solubilization and refolding efficiency. The overall yield of bioactive protein from human growth hormone expressed as bacterial IBs is reported to be around 50%. This is attributed to the capability of novel formulation to disrupt the tertiary structure of the protein while protecting the secondary structure of the protein, thereby reducing the formation of soluble aggregates during refolding. Thus, the formulation can eliminate the need of screening and optimizing various solubilization formulation and will improve the efficiency of recovering bioactive protein from variety of IB aggregates.
Topics: Escherichia coli; Human Growth Hormone; Humans; Inclusion Bodies; Protein Refolding; Protein Structure, Secondary; Protein Structure, Tertiary; Proteins; Solubility; Trifluoroethanol
PubMed: 34798190
DOI: 10.1016/j.ijbiomac.2021.11.068 -
World Journal of Microbiology &... May 2015Papain-like cysteine proteases are widely expressed, fulfill specific functions in extracellular matrix turnover, antigen presentation and processing events, and may... (Review)
Review
Papain-like cysteine proteases are widely expressed, fulfill specific functions in extracellular matrix turnover, antigen presentation and processing events, and may represent viable drug targets for major diseases. In depth and rigorous studies of the potential for these proteins to be targets for drug development require sufficient amounts of protease protein that can be used for both experimental and therapeutic purposes. Escherichia coli was widely used to express papain-like cysteine proteases, but most of those proteases are produced in insoluble inclusion bodies that need solubilizing, refolding, purifying and activating. Refolding is the most critical step in the process of generating active cysteine proteases and the current approaches to refolding include dialysis, dilution and chromatography. Purification is mainly achieved by various column chromatography. Finally, the attained refolded proteases are examined regarding their protease structures and activities.
Topics: Chromatography, Liquid; Cysteine Proteases; Escherichia coli; Inclusion Bodies; Protein Refolding; Recombinant Proteins
PubMed: 25792298
DOI: 10.1007/s11274-015-1804-7