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Organic & Biomolecular Chemistry Jun 2021Fluorescent probes provide an unparalleled opportunity to visualize and quantify dynamic events. Here, we employ a medium-size, cysteine specific coumarin based...
Fluorescent probes provide an unparalleled opportunity to visualize and quantify dynamic events. Here, we employ a medium-size, cysteine specific coumarin based switch-ON fluorescent probe 'L' to track protein unfolding profiles and accessibility of cysteine residues in proteins. It was established that 'L' is highly selective and exhibits no artifact due to interaction with other bystander species. 'L' is able to gauge subtle changes in protein microenvironment and proved to be effective in delineating early unfolding events that are difficult to otherwise discern by classic techniques such as circular dichroism. By solving the X-ray structure of TadA and probing the temperature dependent fluorescence-ON response with native TadA and its cysteine mutants, it was revealed that unfolding occurs in a stage-wise manner and the regions that are functionally important form compact sub-domains and unfold at later stages. Our results assert that probe 'L' serves as an efficient tool to monitor subtle changes in protein structure and can be employed as a generic dye to study processes such as protein unfolding.
Topics: Coumarins; Cysteine; Fluorescent Dyes; Models, Molecular; Molecular Structure; Protein Unfolding; Proteins
PubMed: 34037063
DOI: 10.1039/d1ob00698c -
Methods in Molecular Biology (Clifton,... 2020Differential scanning fluorimetry is useful for a wide variety of applications including characterization of protein function, structure-activity relationships, drug...
Differential scanning fluorimetry is useful for a wide variety of applications including characterization of protein function, structure-activity relationships, drug screening, and optimization of buffer conditions for protein purification, enzyme activity, and crystallization. A limitation of classic differential scanning fluorimetry is its reliance on highly purified protein samples. This limitation is overcome through differential scanning fluorimetry of GFP-tagged proteins (DSF-GTP). DSF-GTP specifically measures the unfolding and aggregation of a target protein fused to GFP through its proximal perturbation effects on GFP fluorescence. As a result of this unique principle, DSF-GTP can specifically measure the thermal stability of a target protein in the presence of other proteins. Additionally, the GFP provides a unique in-assay quality control measure. Here, we describe the workflow, steps, and important considerations for executing a DSF-GTP experiment in a 96-well plate format.
Topics: Calorimetry, Differential Scanning; Fluorescence; Fluorometry; Green Fluorescent Proteins; High-Throughput Screening Assays; Protein Unfolding; Structure-Activity Relationship
PubMed: 31773648
DOI: 10.1007/978-1-0716-0163-1_5 -
Computers in Biology and Medicine Mar 2023Non-covalent intramolecular interactions play a key role in the protein folding process. Aminoacidic mutations or changes in physiological conditions such as pH and/or...
Non-covalent intramolecular interactions play a key role in the protein folding process. Aminoacidic mutations or changes in physiological conditions such as pH and/or temperature variations can compromise intramolecular stability generating misfolding or unfolding proteins with consequent impairment of functionality and the triggering of pathological states. The intramolecular HINT scoring function recently implemented and validated, is proposed as a rapid and sensitive method for the evaluation of different conformational states characterizing destabilization processes. In this work, the stability of Transthyretin, whose denaturation is related to amyloid fibril formation, is evaluated by generating multiple structural mutated models under different pH conditions in comparison with experimental data. These results suggest that the HINT scoring function can be used for an accurate and rapid evaluation and computational prediction of the effects of structural changes on any protein system.
Topics: Prealbumin; Amyloid; Comprehension; Protein Denaturation; Protein Folding
PubMed: 36805224
DOI: 10.1016/j.compbiomed.2023.106667 -
The Journal of Physical Chemistry. B Aug 2021High pressures can be detrimental for protein stability, resulting in unfolding and loss of function. This phenomenon occurs because the unfolding transition is...
High pressures can be detrimental for protein stability, resulting in unfolding and loss of function. This phenomenon occurs because the unfolding transition is accompanied by a decrease in volume, which is typically attributed to the elimination of cavities that are present within the native state as a result of packing defects. We present a novel computational approach that enables the study of pressure unfolding in atomistically detailed protein models in implicit solvent. We include the effect of pressure using a transfer free energy term that allows us to decouple the effect of protein residues and bound water molecules on the volume change upon unfolding. We discuss molecular dynamics simulations results using this protocol for two model proteins, Trp-cage and staphylococcal nuclease (SNase). We find that the volume reduction of bound water is the key energetic term that drives protein denaturation under the effect of pressure, for both Trp-cage and SNase. However, we note differences in unfolding mechanisms between the smaller Trp-cage and the larger SNase protein. Indeed, the unfolding of SNase, but not Trp-cage, is seen to be further accompanied by a reduction in the volume of internal cavities. Our results indicate that, for small peptides, like Trp-cage, pressure denaturation is driven by the increase in solvent accessibility upon unfolding, and the subsequent increase in the number of bound water molecules. For larger proteins, like SNase, the cavities within the native fold act as weak spots, determining the overall resistance to pressure denaturation. Our simulations display a striking agreement with the pressure-unfolding profile experimentally obtained for SNase and represent a promising approach for a computationally efficient and accurate exploration of pressure-induced denaturation of proteins.
Topics: Micrococcal Nuclease; Pressure; Protein Denaturation; Protein Folding; Thermodynamics; Water
PubMed: 34310136
DOI: 10.1021/acs.jpcb.1c04398 -
Biophysical Journal Mar 2020Mechanical processes are involved at many stages of the development of living cells, and often external forces applied to a biomolecule result in its unfolding. Although...
Mechanical processes are involved at many stages of the development of living cells, and often external forces applied to a biomolecule result in its unfolding. Although our knowledge of the unfolding mechanisms and the magnitude of the forces involved has evolved, the role that water molecules play in the mechanical unfolding of biomolecules has not yet been fully elucidated. To this end, we investigated with steered molecular dynamics simulations the mechanical unfolding of dystrophin's spectrin repeat 1 and related the changes in the protein's structure to the ordering of the surrounding water molecules. Our results indicate that upon mechanically induced unfolding of the protein, the solvent molecules become more ordered and increase their average number of hydrogen bonds. In addition, the unfolded structures originating from mechanical pulling expose an increasing amount of the hydrophobic residues to the solvent molecules, and the uncoiled regions adapt a convex surface with a small radius of curvature. As a result, the solvent molecules reorganize around the protein's small protrusions in structurally ordered waters that are characteristic of the so-called "small-molecule regime," which allows water to maintain a high hydrogen bond count at the expense of an increased structural order. We also determined that the response of water to structural changes in the protein is localized to the specific regions of the protein that undergo unfolding. These results indicate that water plays an important role in the mechanically induced unfolding of biomolecules. Our findings may prove relevant to the ever-growing interest in understanding macromolecular crowding in living cells and their effects on protein folding, and suggest that the hydration layer may be exploited as a means for short-range allosteric communication.
Topics: Hydrogen Bonding; Molecular Dynamics Simulation; Protein Folding; Protein Unfolding; Spectrin; Water
PubMed: 32027822
DOI: 10.1016/j.bpj.2020.01.005 -
Proceedings of the National Academy of... Apr 2021Many small proteins move across cellular compartments through narrow pores. In order to thread a protein through a constriction, free energy must be overcome to either...
Many small proteins move across cellular compartments through narrow pores. In order to thread a protein through a constriction, free energy must be overcome to either deform or completely unfold the protein. In principle, the diameter of the pore, along with the effective driving force for unfolding the protein, as well as its barrier to translocation, should be critical factors that govern whether the process proceeds via squeezing, unfolding/threading, or both. To probe this for a well-established protein system, we studied the electric-field-driven translocation behavior of cytochrome (cyt ) through ultrathin silicon nitride (SiN) solid-state nanopores of diameters ranging from 1.5 to 5.5 nm. For a 2.5-nm-diameter pore, we find that, in a threshold electric-field regime of ∼30 to 100 MV/m, cyt is able to squeeze through the pore. As electric fields inside the pore are increased, the unfolded state of cyt is thermodynamically stabilized, facilitating its translocation. In contrast, for 1.5- and 2.0-nm-diameter pores, translocation occurs only by threading of the fully unfolded protein after it transitions through a higher energy unfolding intermediate state at the mouth of the pore. The relative energies between the metastable, intermediate, and unfolded protein states are extracted using a simple thermodynamic model that is dictated by the relatively slow (∼ms) protein translocation times for passing through the nanopore. These experiments map the various modes of protein translocation through a constriction, which opens avenues for exploring protein folding structures, internal contacts, and electric-field-induced deformability.
Topics: Constriction; Cytochromes c; Electricity; Models, Molecular; Nanopores; Protein Folding; Protein Transport; Protein Unfolding; Silicon Compounds; Thermodynamics
PubMed: 33883276
DOI: 10.1073/pnas.2016262118 -
Molecular Cell Sep 2022Oxidative stress conditions can cause ATP depletion, oxidative protein unfolding, and potentially toxic protein aggregation. To alleviate this proteotoxic stress, the...
Oxidative stress conditions can cause ATP depletion, oxidative protein unfolding, and potentially toxic protein aggregation. To alleviate this proteotoxic stress, the highly conserved yeast protein, Get3, switches from its guiding function as an ATP-dependent targeting factor for tail-anchored proteins to its guarding function as an ATP-independent molecular chaperone that prevents irreversible protein aggregation. Here, we demonstrate that activation of Get3's chaperone function follows a tightly orchestrated multi-step process, centered around the redox status of two conserved cysteines, whose reactivity is directly controlled by Get3's nucleotide-binding state. Thiol oxidation causes local unfolding and the transition into chaperone-active oligomers. Vice versa, inactivation requires the reduction of Get3's cysteines followed by ATP-binding, which allows the transfer of bound client proteins to ATP-dependent chaperone systems for their effective refolding. Manipulating this fine-tuned cycle of activation and inactivation in yeast impairs oxidative stress resistance and growth, illustrating the necessity to tightly control Get3's intrinsic chaperone function.
Topics: Adenosine Triphosphatases; Adenosine Triphosphate; Guanine Nucleotide Exchange Factors; Molecular Chaperones; Protein Aggregates; Protein Unfolding; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins
PubMed: 35839781
DOI: 10.1016/j.molcel.2022.06.015 -
Physical Chemistry Chemical Physics :... Jul 2022A complete thermodynamic description of protein-ligand binding includes parameters related to pressure and temperature. The changes in the protein volume and...
A complete thermodynamic description of protein-ligand binding includes parameters related to pressure and temperature. The changes in the protein volume and compressibility upon binding a ligand are pressure-related parameters that are often neglected due to the lack of routine methods for their determination. Fluorescent pressure shift assay (FPSA) is based on pressure-induced protein unfolding and its stabilization by a ligand and offers a universal approach to determine protein-ligand binding volumes. Extremely high pressures are required to unfold most proteins and protein-ligand complexes. Thus, guanidinium hydrochloride (GdmHCl) is used as a protein-destabilizing agent. We determined that GdmHCl unfolds carbonic anhydrase isoforms in a different pathway, but the destabilization effect is linear in a particular concentration range. We developed a concept for the FPSA experiment, where both - the ligand and GdmHCl - concentrations are varied. This approach enabled us to determine protein-ligand binding volumes that otherwise would be impossible due to the equipment-unreachable pressures of protein unfolding.
Topics: Guanidine; Ligands; Protein Denaturation; Protein Unfolding; Proteins; Thermodynamics
PubMed: 35802138
DOI: 10.1039/d2cp01046a -
International Journal of Biological... Jun 2021The thermal unfolding of the copper redox protein azurin was studied in the presence of four different amino acid-based ionic liquids (ILs), all of which have...
The thermal unfolding of the copper redox protein azurin was studied in the presence of four different amino acid-based ionic liquids (ILs), all of which have tetramethylguanidium as cation. The anionic amino acid includes two with alcohol side chains, serine and threonine, and two with carboxylic acids, aspartate and glutamate. Control experiments showed that amino acids alone do not significantly change protein stability and pH changes anticipated by the amino acid nature have only minor effects on the protein. With the ILs, the protein is destabilized and the melting temperature is decreased. The two ILs with alcohol side chains strongly destabilize the protein while the two ILs with acid side chains have weaker effects. Unfolding enthalpy (ΔH) and entropy (ΔS) values, derived from fits of the unfolding data, show that some ILs increase ΔHwhile others do not significantly change this value. All ILs, however, increase ΔS. MD simulations of both the folded and unfolded protein conformations in the presence of the ILs provide insight into the different IL-protein interactions and how they affect the ΔH values. The simulations also confirm that the ILs increase the unfolded state entropies which can explain the increased ΔS values.
Topics: Amino Acids; Anions; Azurin; Cations; Entropy; Hydrogen-Ion Concentration; Hydrophobic and Hydrophilic Interactions; Imidazoles; Ionic Liquids; Methylguanidine; Molecular Dynamics Simulation; Protein Stability; Protein Structure, Secondary; Protein Unfolding; Transition Temperature
PubMed: 33744247
DOI: 10.1016/j.ijbiomac.2021.03.090 -
Molecules (Basel, Switzerland) Mar 2022Frataxin (FXN) is a protein involved in storage and delivery of iron in the mitochondria. Single-point mutations in the gene lead to reduced production of functional...
Frataxin (FXN) is a protein involved in storage and delivery of iron in the mitochondria. Single-point mutations in the gene lead to reduced production of functional frataxin, with the consequent dyshomeostasis of iron. FXN variants are at the basis of neurological impairment (the Friedreich's ataxia) and several types of cancer. By using altruistic metadynamics in conjunction with the maximal constrained entropy principle, we estimate the change of free energy in the protein unfolding of frataxin and of some of its pathological mutants. The sampled configurations highlight differences between the wild-type and mutated sequences in the stability of the folded state. In partial agreement with thermodynamic experiments, where most of the analyzed variants are characterized by lower thermal stability compared to wild type, the D104G variant is found with a stability comparable to the wild-type sequence and a lower water-accessible surface area. These observations, obtained with the new approach we propose in our work, point to a functional switch, affected by single-point mutations, of frataxin from iron storage to iron release. The method is suitable to investigate wide structural changes in proteins in general, after a proper tuning of the chosen collective variable used to perform the transition.
Topics: Friedreich Ataxia; Humans; Iron-Binding Proteins; Protein Unfolding; Thermodynamics; Frataxin
PubMed: 35335316
DOI: 10.3390/molecules27061955