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Proceedings of the National Academy of... Mar 2021Single amino acid mutations provide quantitative insight into the energetics that underlie the dynamics and folding of membrane proteins. Chemical denaturation is the...
Single amino acid mutations provide quantitative insight into the energetics that underlie the dynamics and folding of membrane proteins. Chemical denaturation is the most widely used assay and yields the change in unfolding free energy (ΔΔ). It has been applied to >80 different residues of bacteriorhodopsin (bR), a model membrane protein. However, such experiments have several key limitations: 1) a nonnative lipid environment, 2) a denatured state with significant secondary structure, 3) error introduced by extrapolation to zero denaturant, and 4) the requirement of globally reversible refolding. We overcame these limitations by reversibly unfolding local regions of an individual protein with mechanical force using an atomic-force-microscope assay optimized for 2 μs time resolution and 1 pN force stability. In this assay, bR was unfolded from its native bilayer into a well-defined, stretched state. To measure ΔΔ, we introduced two alanine point mutations into an 8-amino-acid region at the C-terminal end of bR's G helix. For each, we reversibly unfolded and refolded this region hundreds of times while the rest of the protein remained folded. Our single-molecule-derived ΔΔ for mutant L223A (-2.3 ± 0.6 kcal/mol) quantitatively agreed with past chemical denaturation results while our ΔΔ for mutant V217A was 2.2-fold larger (-2.4 ± 0.6 kcal/mol). We attribute the latter result, in part, to contact between Val and a natively bound squalene lipid, highlighting the contribution of membrane protein-lipid contacts not present in chemical denaturation assays. More generally, we established a platform for determining ΔΔ for a fully folded membrane protein embedded in its native bilayer.
Topics: Amino Acid Substitution; Bacteriorhodopsins; Halobacterium salinarum; Lipid Bilayers; Microscopy, Atomic Force; Point Mutation; Protein Folding; Single Molecule Imaging; Thermodynamics
PubMed: 33753487
DOI: 10.1073/pnas.2020083118 -
FEBS Letters Jan 2014Today, proteins are typically overexpressed using solubility-enhancing fusion tags that allow for affinity chromatographic purification and subsequent removal by... (Review)
Review
Today, proteins are typically overexpressed using solubility-enhancing fusion tags that allow for affinity chromatographic purification and subsequent removal by site-specific protease cleavage. In this review, we present an alternative approach to protein production using fusion partners specifically designed to accumulate in insoluble inclusion bodies. The strategy is appropriate for the mass production of short peptides, intrinsically disordered proteins, and proteins that can be efficiently refolded in vitro. There are many fusion protein systems now available for insoluble expression: TrpLE, ketosteroid isomerase, PurF, and PagP, for example. The ideal fusion partner is effective at directing a wide variety of target proteins into inclusion bodies, accumulates in large quantities in a highly pure form, and is readily solubilized and purified in commonly used denaturants. Fusion partner removal under denaturing conditions is biochemically challenging, requiring harsh conditions (e.g., cyanogen bromide in 70% formic acid) that can result in unwanted protein modifications. Recent advances in metal ion-catalyzed peptide bond cleavage allow for more mild conditions, and some methods involving nickel or palladium will likely soon appear in more biological applications.
Topics: Escherichia coli; Gene Expression; Genetic Engineering; Humans; Inclusion Bodies; Protein Denaturation; Recombinant Fusion Proteins
PubMed: 24076468
DOI: 10.1016/j.febslet.2013.09.028 -
Proceedings of the National Academy of... Oct 2012We use molecular simulations using a coarse-grained model to map the folding landscape of Green Fluorescent Protein (GFP), which is extensively used as a marker in cell...
We use molecular simulations using a coarse-grained model to map the folding landscape of Green Fluorescent Protein (GFP), which is extensively used as a marker in cell biology and biotechnology. Thermal and Guanidinium chloride (GdmCl) induced unfolding of a variant of GFP, without the chromophore, occurs in an apparent two-state manner. The calculated midpoint of the equilibrium folding in GdmCl, taken into account using the Molecular Transfer Model (MTM), is in excellent agreement with the experiments. The melting temperatures decrease linearly as the concentrations of GdmCl and urea are increased. The structural features of rarely populated equilibrium intermediates, visible only in free energy profiles projected along a few order parameters, are remarkably similar to those identified in a number of ensemble experiments in GFP with the chromophore. The excellent agreement between simulations and experiments show that the equilibrium intermediates are stabilized by the chromophore. Folding kinetics, upon temperature quench, show that GFP first collapses and populates an ensemble of compact structures. Despite the seeming simplicity of the equilibrium folding, flux to the native state flows through multiple channels and can be described by the kinetic partitioning mechanism. Detailed analysis of the folding trajectories show that both equilibrium and several kinetic intermediates, including misfolded structures, are sampled during folding. Interestingly, the intermediates characterized in the simulations coincide with those identified in single molecule pulling experiments. Our predictions, amenable to experimental tests, show that MTM is a practical way to simulate the effect of denaturants on the folding of large proteins.
Topics: Green Fluorescent Proteins; Guanidine; Hot Temperature; Kinetics; Models, Molecular; Protein Denaturation; Protein Folding; Protein Structure, Secondary
PubMed: 22778437
DOI: 10.1073/pnas.1201808109 -
Life (Basel, Switzerland) Jul 2021The conformational stability of globular proteins is strongly influenced by the addition to water of different co-solutes. Some of the latter destabilize the native...
The conformational stability of globular proteins is strongly influenced by the addition to water of different co-solutes. Some of the latter destabilize the native state, while others stabilize it. It is emerging that stabilizing agents are able to counteract the action of destabilizing agents. We have already provided experimental evidence that this counteraction is a general phenomenon and offered a rationalization. In the present work, we show that four different sugars, namely fructose, glucose, sucrose, and trehalose, counteract the effect of urea, tetramethylurea, sodium perchlorate, guanidinium chloride, and guanidinium thiocyanate despite the chemical and structural differences of those destabilizing agents. The rationalization we provide is as follows: (a) the solvent-excluded volume effect, a purely entropic effect, stabilizes the native state, whose solvent-accessible surface area is smaller than the one of denatured conformations; (b) the magnitude of the solvent-excluded volume effect increases markedly in ternary solutions because the experimental density of such solutions is larger than that of pure water.
PubMed: 34357025
DOI: 10.3390/life11070652 -
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 -
Biochemistry May 2022The structure of the first ubiquitin-associated domain from HHR23A, UBA(1), was determined by X-ray crystallography at a 1.60 Å resolution, and its stability, folding...
The structure of the first ubiquitin-associated domain from HHR23A, UBA(1), was determined by X-ray crystallography at a 1.60 Å resolution, and its stability, folding kinetics, and residual structure under denaturing conditions have been investigated. The concentration dependence of thermal denaturation and size-exclusion chromatography indicate that UBA(1) is monomeric. Guanidine hydrochloride (GdnHCl) denaturation experiments reveal that the unfolding free energy, Δ°'(HO), of UBA(1) is 2.4 kcal mol. Stopped-flow folding kinetics indicates sub-millisecond folding with only proline isomerization phases detectable at 25 °C. The full folding kinetics are observable at 4 °C, yielding a folding rate constant, , in the absence of a denaturant of 13,000 s and a Tanford β-value of 0.80, consistent with a compact transition state. Evaluation of the secondary structure via circular dichroism shows that the residual helical structure in the denatured state is replaced by polyproline II structure as the GdnHCl concentration increases. Analysis of NMR secondary chemical shifts for backbone NH, CO, and Cα atoms between 4 and 7 M GdnHCl shows three islands of residual helical secondary structure that align in sequence with the three native-state helices. Extrapolation of the NMR data to 0 M GdnHCl demonstrates that helical structure would populate to 17-33% in the denatured state under folding conditions. Comparison with NMR data for a peptide corresponding to helix 1 indicates that this helix is stabilized by transient tertiary interactions in the denatured state of UBA(1). The high helical content in the denatured state, which is enhanced by transient tertiary interactions, suggests a diffusion-collision folding mechanism.
Topics: Circular Dichroism; DNA; DNA Repair; Guanidine; Humans; Kinetics; Protein Denaturation; Protein Folding; Thermodynamics
PubMed: 35430812
DOI: 10.1021/acs.biochem.2c00011 -
PloS One 2014Extensive and intensive studies on the unfolding of proteins require appropriate theoretical model and parameter to clearly illustrate the feature and characteristic of...
BACKGROUND
Extensive and intensive studies on the unfolding of proteins require appropriate theoretical model and parameter to clearly illustrate the feature and characteristic of the unfolding system. Over the past several decades, four approaches have been proposed to describe the interaction between proteins and denaturants, but some ambiguity and deviations usually occur in the explanation of the experimental data.
METHODOLOGY/PRINCIPAL FINDINGS
In this work, a theoretical model was presented to show the dependency of the residual activity ratio of the proteins on the molar denaturant concentration. Through the characteristic unfolding parameters ki and Δmi in this model, the distribution, transition and thermodynamic stability of protein conformations during the unfolding process can be quantitatively described. This model was tested with the two-state unfolding of bovine heart cytochrome c and the three-state unfolding of hen egg white lysozyme induced by both guanidine hydrochloride and urea, the four-state unfolding of bovine carbonic anhydrase b induced by guanidine hydrochloride and the unfolding of some other proteins induced by denaturants. The results illustrated that this model could be used accurately to reveal the distribution and transition of protein conformations in the presence of different concentrations of denaturants and to evaluate the unfolding tendency and thermodynamic stability of different conformations. In most denaturant-induced unfolding of proteins, the unfolding became increasingly hard in next transition step and the proteins became more unstable as they attained next successive stable conformation.
CONCLUSIONS/SIGNIFICANCE
This work presents a useful method for people to study the unfolding of proteins and may be used to describe the unfolding and refolding of other biopolymers induced by denaturants, inducers, etc.
Topics: Animals; Carbonic Anhydrases; Cattle; Chickens; Cytochromes c; Guanidine; Muramidase; Myocardium; Protein Conformation; Protein Denaturation; Protein Unfolding; Regression Analysis; Thermodynamics; Urea
PubMed: 24603868
DOI: 10.1371/journal.pone.0091129 -
International Journal of Molecular... Sep 2018Near-infrared fluorescent proteins (NIR FPs) based on the complexes of bacterial phytochromes with their natural biliverdin chromophore are widely used as genetically...
Near-infrared fluorescent proteins (NIR FPs) based on the complexes of bacterial phytochromes with their natural biliverdin chromophore are widely used as genetically encoded optical probes for visualization of cellular processes and deep-tissue imaging of cells and organs in living animals. In this work, we show that the steady-state and kinetic dependencies of the various spectral characteristics of iRFP713, developed from the bacterial phytochrome BphP2 and recorded at protein unfolding induced by guanidine hydrochloride (GdnHCl), guanidine thiocyanate (GTC), and urea, differ substantially. A study of the unfolding of three single-tryptophan mutant forms of iRFP713 expectedly revealed that protein unfolding begins with the dissociation of the native dimer, while the monomers remain compact. A further increase in the denaturant concentration leads to the formation of an intermediate state of iRFP713 having hydrophobic areas exposed on the protein surface (I). The total surface charge of iRFP713 (pI 5.86) changes from negative to positive with an increase in the concentration of GdnHCl and GTC because the negative charge of glutamic and aspartic acids is neutralized by forming salt bridges between the carboxyl groups and GdnH⁺ ions and because the guanidinium cations bind to amide groups of glutamines and asparagines. The coincidence of both the concentration of the denaturants at which the intermediate state of iRFP713 accumulates and the concentration of GdnH⁺ ions at which the neutralization of the surface charge of the protein in this state is ensured results in strong protein aggregation. This is evidently realized by iRFP713 unfolding by GTC. At the unfolding of the protein by GdnHCl, an intermediate state is populated at higher denaturant concentrations and a strong aggregation is not observed. As expected, protein aggregates are not formed in the presence of the urea. The aggregation of the protein upon neutralization of the charge on the macromolecule surface is the main indicator of the intermediate state of protein. The unfolded state of iRFP713, whose formation is accompanied by a significant decrease in the parameter , was found to have a different residual structure in the denaturants used.
Topics: Guanidine; Guanidines; Kinetics; Luminescent Proteins; Protein Aggregates; Protein Denaturation; Protein Folding; Protein Unfolding; Thiocyanates
PubMed: 30223568
DOI: 10.3390/ijms19092776 -
Journal of the American Chemical Society Sep 2016Chemical denaturants are the most commonly used agents for unfolding proteins and are thought to act by better solvating the unfolded state. Improved solvation is...
Chemical denaturants are the most commonly used agents for unfolding proteins and are thought to act by better solvating the unfolded state. Improved solvation is expected to lead to an expansion of unfolded chains with increasing denaturant concentration, providing a sensitive probe of the denaturant action. However, experiments have so far yielded qualitatively different results concerning the effects of chemical denaturation. Studies using Förster resonance energy transfer (FRET) and other methods found an increase in radius of gyration with denaturant concentration, but most small-angle X-ray scattering (SAXS) studies found no change. This discrepancy therefore challenges our understanding of denaturation mechanism and more generally the accuracy of these experiments as applied to unfolded or disordered proteins. Here, we use all-atom molecular simulations to investigate the effect of urea and guanidinium chloride on the structure of the intrinsically disordered protein ACTR, which can be studied by experiment over a wide range of denaturant concentration. Using unbiased molecular simulations with a carefully calibrated denaturant model, we find that the protein chain indeed swells with increasing denaturant concentration. This is due to the favorable association of urea or guanidinium chloride with the backbone of all residues and with the side-chains of almost all residues, with denaturant-water transfer free energies inferred from this association in reasonable accord with experimental estimates. Interactions of the denaturants with the backbone are dominated by hydrogen bonding, while interactions with side-chains include other contributions. By computing FRET efficiencies and SAXS intensities at each denaturant concentration, we show that the simulation trajectories are in accord with both experiments on this protein, demonstrating that there is no fundamental inconsistency between the two types of experiment. Agreement with experiment also supports the picture of chemical denaturation described in our simulations, driven by weak association of denaturant with the protein. Our simulations support some assumptions needed for each experiment to accurately reflect changes in protein size, namely, that the commonly used FRET chromophores do not qualitatively alter the results and that possible effects such as preferential solvent partitioning into the interior of the chain do not interfere with the determination of radius of gyration from the SAXS experiments.
Topics: Intrinsically Disordered Proteins; Molecular Dynamics Simulation; Protein Conformation; Protein Denaturation; Urea
PubMed: 27583687
DOI: 10.1021/jacs.6b05443 -
Protein Science : a Publication of the... Mar 2016Determining the energetics of the unfolded state of a protein is essential for understanding the folding mechanics of ordered proteins and the structure-function...
Determining the energetics of the unfolded state of a protein is essential for understanding the folding mechanics of ordered proteins and the structure-function relation of intrinsically disordered proteins. Here, we adopt a coil-globule transition theory to develop a general scheme to extract interaction and free energy information from single-molecule fluorescence resonance energy transfer spectroscopy. By combining protein stability data, we have determined the free energy difference between the native state and the maximally collapsed denatured state in a number of systems, providing insight on the specific/nonspecific interactions in protein folding. Both the transfer and binding models of the denaturant effects are demonstrated to account for the revealed linear dependence of inter-residue interactions on the denaturant concentration, and are thus compatible under the coil-globule transition theory to further determine the dimension and free energy of the conformational ensemble of the unfolded state. The scaling behaviors and the effective θ-state are also discussed.
Topics: Fluorescence Resonance Energy Transfer; Protein Conformation; Protein Denaturation; Protein Folding; Protein Stability; Proteins; Thermodynamics
PubMed: 26683260
DOI: 10.1002/pro.2865