-
Biochemical and Biophysical Research... Apr 2021The mechanism for protein stabilization or destabilization has long been an open quest. In the present study, we have studied the interactions between amino acids and...
The mechanism for protein stabilization or destabilization has long been an open quest. In the present study, we have studied the interactions between amino acids and guanidinium (Gdm)/ammonium (NH) ions by using low field nuclear magnetic resonance (LF-NMR), where Gdm and NH are denaturant and stabilizer for proteins, respectively. It shows that Gdm favors to bind to the thiol group or the hydroxyl group on the side chain but weakly interacts with the α-carboxyl group. In contrast, NH prefers to bind to the α-carboxyl group but slightly interacts with the thiol group or the hydroxyl group on the side chain of amino acids. HNMR reveals the hydrogen bonding between NH and the α-carboxyl group, which is not involved in the interactions between Gdm and cysteine. Our study demonstrates that the strong interactions between the denaturant and the sulfur atom or the disulfide bond promote the direct binding of the denaturant toward proteins, leading to the destabilization.
Topics: Amino Acids; Ammonium Chloride; Cations; Guanidine; Hydrogen; Protein Stability; Proton Magnetic Resonance Spectroscopy; Solutions
PubMed: 33631673
DOI: 10.1016/j.bbrc.2021.02.017 -
Cold Spring Harbor Protocols Nov 2021Alkaline agarose gels are run at high pH, which causes each thymine and guanine residue to lose a proton and thus prevents the formation of hydrogen bonds with their...
Alkaline agarose gels are run at high pH, which causes each thymine and guanine residue to lose a proton and thus prevents the formation of hydrogen bonds with their adenine and cytosine partners. The denatured DNA is maintained in a single-stranded state and migrates through an alkaline agarose gel as a function of its size. Other denaturants such as formamide and urea do not work well because they cause the agarose to become rubbery.
Topics: Cytosine; DNA; Electrophoresis, Agar Gel; Sepharose
PubMed: 34725171
DOI: 10.1101/pdb.prot100438 -
Current Protocols in Protein Science Nov 2018Electrophoresis is used to separate complex mixtures of proteins (e.g., from cells, subcellular fractions, column fractions, or immunoprecipitates), to investigate...
Electrophoresis is used to separate complex mixtures of proteins (e.g., from cells, subcellular fractions, column fractions, or immunoprecipitates), to investigate subunit composition, track post-translational modifications, and verify identity and homogeneity of protein samples. It can also serve to purify proteins for use in further applications. In polyacrylamide gel electrophoresis, proteins migrate in response to an electrical field through pores in a polyacrylamide gel matrix; pore size decreases with increasing acrylamide concentration. Nondenaturing or "native" electrophoresis-i.e., electrophoresis in the absence of denaturants such as detergents and urea-is an often-overlooked technique for determining the native size, subunit structure, and optimal separation of a protein. Because mobility depends on the size, shape, and intrinsic charge of the protein, nondenaturing electrophoresis provides a set of separation parameters distinctly different from mainly size-dependent denaturing sodium dodecyl sulfate-polyacrylamide gel electrophoresis and charge-dependent isoelectric focusing. Two protocols are presented below. Continuous PAGE is highly flexible, permitting cationic and anionic electrophoresis over a full range of pH. The discontinuous procedure is limited to proteins negatively charged at neutral pH but provides high resolution for accurate size calibration.
Topics: Electrophoresis, Polyacrylamide Gel; Proteins
PubMed: 30091848
DOI: 10.1002/cpps.73 -
Current Protocols in Protein Science Nov 2014Heterologous expression of recombinant proteins in E. coli often results in the formation of insoluble and inactive protein aggregates, commonly referred to as inclusion... (Review)
Review
Heterologous expression of recombinant proteins in E. coli often results in the formation of insoluble and inactive protein aggregates, commonly referred to as inclusion bodies. To obtain the native (i.e., correctly folded) and hence active form of the protein from such aggregates, four steps are usually followed: (1) the cells are lysed, (2) the cell wall and outer membrane components are removed, (3) the aggregates are solubilized (or extracted) with strong protein denaturants, and (4) the solubilized, denatured proteins are folded with concomitant oxidation of reduced cysteine residues into the correct disulfide bonds to obtain the native protein. This unit features three different approaches to the final step of protein folding and purification. In the first, guanidine·HCl is used as the denaturant, after which the solubilized protein is folded (before purification) in an "oxido-shuffling" buffer system to increase the rate of protein oxidation. In the second, acetic acid is used to solubilize the protein, which is then partially purified by gel filtration before folding; the protein is then folded and oxidized by simple dialysis against water. Thirdly, folding and purification of a fusion protein using metal-chelate affinity chromatography are described.
Topics: Animals; Escherichia coli; Guanidine; Humans; Inclusion Bodies; Protein Denaturation; Protein Refolding; Recombinant Proteins
PubMed: 25367010
DOI: 10.1002/0471140864.ps0605s78 -
The Journal of Physical Chemistry. B Jun 2023The denaturation of DNA is a critical process in biology and has many biotechnological applications. We investigated the compaction of locally denatured DNA by a...
The denaturation of DNA is a critical process in biology and has many biotechnological applications. We investigated the compaction of locally denatured DNA by a chemical denaturation agent, dimethyl sulfoxide (DMSO), using magnetic tweezers (MTs), atomic force microscopy (AFM), and dynamic light scattering (DLS). Our results show that DMSO not only is capable of denaturing DNA but also able to compact DNA directly. When the DMSO concentration is above 10%, DNA condensation occurs due to the reduction in the persistence length of DNA and excluded volume interactions. Meanwhile, locally denatured DNA is easily condensed by divalent cations, such as magnesium ions (Mg), contrasting with no native DNA condensation by the classical divalent cations. Specifically, the addition of more than 3 mM Mg to a 5% DMSO solution leads to DNA condensation. The critical condensing force () increases from 6.4 to 9.5 pN when the Mg concentration grows from 3 to 10 mM. However, decreases gradually with a further increase in Mg concentration. For 3% DMSO solution, above 30 mM Mg is needed to compact DNA and a weaker condensing force was measured. With increasing Mg concentration, the morphology of the DMSO partially denatured DNA complex changes from loosely random coils to a dense network structure, even forming a spherical condensation nucleus, and finally to a partially disintegrated network. These findings show that the elasticity of DNA plays an important role in its denaturation and condensation behavior.
Topics: Cations, Divalent; Dimethyl Sulfoxide; DNA; Cell Nucleus; Elasticity; Cations
PubMed: 37205854
DOI: 10.1021/acs.jpcb.3c01858 -
Nature Protocols May 2022DNA fluorescence in situ hybridization (FISH) has been a central technique in advancing our understanding of how chromatin is organized within the nucleus. With the... (Review)
Review
DNA fluorescence in situ hybridization (FISH) has been a central technique in advancing our understanding of how chromatin is organized within the nucleus. With the increasing resolution offered by super-resolution microscopy, the optimal maintenance of chromatin structure within the nucleus is essential for accuracy in measurements and interpretation of data. However, standard 3D-FISH requires potentially destructive heat denaturation in the presence of chaotropic agents such as formamide to allow access to the DNA strands for labeled FISH probes. To avoid the need to heat-denature, we developed Resolution After Single-strand Exonuclease Resection (RASER)-FISH, which uses exonuclease digestion to generate single-stranded target DNA for efficient probe binding over a 2 d process. Furthermore, RASER-FISH is easily combined with immunostaining of nuclear proteins or the detection of RNAs. Here, we provide detailed procedures for RASER-FISH in mammalian cultured cells to detect single loci, chromatin tracks and topologically associating domains with conventional and super-resolution 3D structured illumination microscopy. Moreover, we provide a validation and characterization of our method, demonstrating excellent preservation of chromatin structure and nuclear integrity, together with improved hybridization efficiency, compared with classic 3D-FISH protocols.
Topics: Animals; Cell Nucleus; Chromatin; DNA; Exonucleases; In Situ Hybridization, Fluorescence; Interphase; Mammals
PubMed: 35379945
DOI: 10.1038/s41596-022-00685-8 -
Methods in Molecular Biology (Clifton,... 2022Recombinant protein expression in E. coli often induces the expressed protein to accumulate in insoluble aggregates, named inclusion bodies (IBs), that represent easy to...
Recombinant protein expression in E. coli often induces the expressed protein to accumulate in insoluble aggregates, named inclusion bodies (IBs), that represent easy to isolate, highly pure protein reservoirs. IBs can be solubilized by denaturing agents but this procedure requires, for complex globular proteins, a refolding step that can be challenging. However, the lack of cooperatively folded tertiary structure in intrinsically disordered proteins (IDP) makes them ideal candidates for this purification strategy. Given the wide abundance of IDPs, their relevance in many disease areas and the numerous IDP-associated biological functions, the interest in this class of proteins has increased substantially over the last decade. Here we present a broad and versatile method for the production and isolation of IDPs from inclusion bodies under denaturant conditions that overcomes the challenges associated with the propensity of these sequences to precipitate from solution and becoming proteolytically degraded.
Topics: Escherichia coli; Inclusion Bodies; Intrinsically Disordered Proteins; Recombinant Proteins
PubMed: 35089568
DOI: 10.1007/978-1-0716-1859-2_21 -
Methods in Molecular Biology (Clifton,... 2023Inclusion bodies (IB) are dense insoluble aggregates of mostly misfolded polypeptides that usually result from recombinant protein overexpression. IB formation has been...
Inclusion bodies (IB) are dense insoluble aggregates of mostly misfolded polypeptides that usually result from recombinant protein overexpression. IB formation has been observed in protein expression systems such as E. coli, yeast, and higher eukaryotes. To recover soluble recombinant proteins in their native state, IB are commonly first solubilized with a high concentration of denaturant. This is followed by concurrent denaturant removal or reduction and a transition into a refolding-favorable chemical environment to facilitate the refolding of solubilized protein to its native state. Due to the high concentration of denaturant used, conventional refolding approaches can result in dilute products and are buffer inefficient. To circumvent the limitations of conventional refolding approaches, a temperature-based refolding approach which combines a low concentration of denaturant (0.5 M guanidine hydrochloride, GdnHCl) with a high temperature (95 °C) during solubilization was proposed. In this chapter, we describe a temperature-based refolding approach for the recovery of core streptavidin (cSAV) from IB. Through the temperature-based approach, intensification was achieved through the elimination of a concentration step which would be required by a dilution approach and through a reduction in buffer volumes required for dilution or denaturant removal. High-temperature treatment during solubilization may have also resulted in the denaturation and aggregation of undesired host-cell proteins, which could then be removed through a centrifugation step resulting in refolded cSAV of high purity without the need for column purification. Refolded cSAV was characterized by biotin-binding assay and SDS-PAGE, while purity was determined by RP-HPLC.
Topics: Temperature; Escherichia coli; Recombinant Proteins; Hot Temperature; Inclusion Bodies; Protein Folding; Protein Refolding
PubMed: 36656525
DOI: 10.1007/978-1-0716-2930-7_13 -
Biochemistry Sep 2016Stabilizing the folded state of metastable and/or aggregation-prone proteins through exogenous ligand binding is an appealing strategy for decreasing disease pathologies...
Stabilizing the folded state of metastable and/or aggregation-prone proteins through exogenous ligand binding is an appealing strategy for decreasing disease pathologies caused by protein folding defects or deleterious kinetic transitions. Current methods of examining binding of a ligand to these marginally stable native states are limited because protein aggregation typically interferes with analysis. Here, we describe a rapid method for assessing the kinetic stability of folded proteins and monitoring the effects of ligand stabilization for both intrinsically stable proteins (monomers, oligomers, and multidomain proteins) and metastable proteins (e.g., low Tm) that uses a new GroEL chaperonin-based biolayer interferometry (BLI) denaturant pulse platform. A kinetically controlled denaturation isotherm is generated by exposing a target protein, immobilized on a BLI biosensor, to increasing denaturant concentrations (urea or GuHCl) in a pulsatile manner to induce partial or complete unfolding of the attached protein population. Following the rapid removal of the denaturant, the extent of hydrophobic unfolded/partially folded species that remains is detected by an increased level of GroEL binding. Because this kinetic denaturant pulse is brief, the amplitude of binding of GroEL to the immobilized protein depends on the duration of the exposure to the denaturant, the concentration of the denaturant, wash times, and the underlying protein unfolding-refolding kinetics; fixing all other parameters and plotting the GroEL binding amplitude versus denaturant pulse concentration result in a kinetically controlled denaturation isotherm. When folding osmolytes or stabilizing ligands are added to the immobilized target proteins before and during the denaturant pulse, the diminished population of unfolded/partially folded protein manifests as a decreased level of GroEL binding and/or a marked shift in these kinetically controlled denaturation profiles to higher denaturant concentrations. This particular platform approach can be used to identify small molecules and/or solution conditions that can stabilize or destabilize thermally stable proteins, multidomain proteins, oligomeric proteins, and, most importantly, aggregation-prone metastable proteins.
Topics: Biosensing Techniques; Chaperonin 60; Kinetics; Ligands; Protein Denaturation; Protein Folding; Proteins; Thermodynamics
PubMed: 27505032
DOI: 10.1021/acs.biochem.6b00293 -
European Biophysics Journal : EBJ Apr 2023Due to misincorporation during gene replication, the accuracy of the gene expression is often compromised. This results in a mismatch or defective pair in the DNA...
Due to misincorporation during gene replication, the accuracy of the gene expression is often compromised. This results in a mismatch or defective pair in the DNA molecule (James et al. 2016). Here, we present our study of the stability of DNA with defects in the thermal and force ensembles. We consider DNA with a different number of defects from 2to16 and study how the denaturation process differs in both ensembles. Using a statistical model, we calculate the melting point of the DNA chain in both the ensemble. Our findings display different manifestations of DNA denaturation in thermal and force ensembles. While the DNA with defects denatures at a lower temperature than the intact DNA, the point from which the DNA is pulled is important in force ensemble.
Topics: Base Pairing; Nucleic Acid Conformation; DNA; Nucleic Acid Denaturation; Temperature
PubMed: 37249617
DOI: 10.1007/s00249-023-01659-8