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Nature Chemical Biology Feb 2023Detailed understanding of the mechanism by which Hsp70 chaperones protect cells against protein aggregation is hampered by the lack of a comprehensive characterization...
Detailed understanding of the mechanism by which Hsp70 chaperones protect cells against protein aggregation is hampered by the lack of a comprehensive characterization of the aggregates, which are typically heterogeneous. Here we designed a reporter chaperone substrate, MLucV, composed of a stress-labile luciferase flanked by stress-resistant fluorescent domains, which upon denaturation formed a discrete population of small aggregates. Combining Förster resonance energy transfer and enzymatic activity measurements provided unprecedented details on the aggregated, unfolded, Hsp70-bound and native MLucV conformations. The Hsp70 mechanism first involved ATP-fueled disaggregation and unfolding of the stable pre-aggregated substrate, which stretched MLucV beyond simply unfolded conformations, followed by native refolding. The ATP-fueled unfolding and refolding action of Hsp70 on MLucV aggregates could accumulate native MLucV species under elevated denaturing temperatures highly adverse to the native state. These results unambiguously exclude binding and preventing of aggregation from the non-equilibrium mechanism by which Hsp70 converts stable aggregates into metastable native proteins.
Topics: Protein Folding; HSP70 Heat-Shock Proteins; Molecular Chaperones; Luciferases; Adenosine Triphosphate; Protein Denaturation; Protein Unfolding
PubMed: 36266349
DOI: 10.1038/s41589-022-01162-9 -
Journal of the American Society For... Oct 2023Studies of protein folding often involve offline experimental methods such as titrating protein samples with denaturants or equilibrating them in the presence of...
Studies of protein folding often involve offline experimental methods such as titrating protein samples with denaturants or equilibrating them in the presence of denaturants. Here, we demonstrate an online analytical approach in which the protein structure is perturbed by a pH ramp evoked by immobilized lipase-catalyzed ester hydrolysis. Changes in the tertiary structure of the protein in response to a pH ramp (from approximately 6.3 to 2.8) are monitored using electrospray ionization mass spectrometry and spectrofluorometry. Interestingly, we discovered a side reaction of ammonium and formate leading to the production of cyanide that occurred during the ionization process. We also found that only certain protein analytes were bound to the formed cyanide species. Nevertheless, this problem was readily overcome by carefully selecting a specific ester substrate. Overall, the alterations in the charge-state distribution and fluorescence intensity─caused by the lipase-induced pH ramp─reveal conformational transitions in different proteins. In line with previous reports, the acid-induced denaturation of holo-myoglobin occurs through a two-step mechanism, which is supported by identification of protein-unfolding intermediates and the loss of noncovalent protein ligand (heme). The results─obtained using the developed catalytic method─are also consistent with the results of equilibrium-based experiments, while sample preparation steps are substantially reduced. The proposed approach simplifies the identification of the pH range that has the greatest impact on the protein structure. Thus, it has the potential to be a useful tool for studying protein conformational transitions in the course of pH changes.
Topics: Lipase; Hydrolysis; Protein Denaturation; Protein Unfolding; Protein Folding; Myoglobin; Hydrogen-Ion Concentration; Cyanides
PubMed: 37620995
DOI: 10.1021/jasms.3c00224 -
Advanced Biology Dec 2023Single molecule techniques are particularly well suited for investigating the processes of protein folding and chaperone assistance. However, current assays provide only...
Single molecule techniques are particularly well suited for investigating the processes of protein folding and chaperone assistance. However, current assays provide only a limited perspective on the various ways in which the cellular environment can influence the folding pathway of a protein. In this study, a single molecule mechanical interrogation assay is developed and used to monitor protein unfolding and refolding within a cytosolic solution. This allows to test the cumulative topological effect of the cytoplasmic interactome on the folding process. The results reveal a stabilization against forced unfolding for partial folds, which are attributed to the protective effect of the cytoplasmic environment against unfolding and aggregation. This research opens the possibility of conducting single molecule molecular folding experiments in quasi-biological environments.
Topics: Protein Unfolding; Protein Folding
PubMed: 37409427
DOI: 10.1002/adbi.202300105 -
Accounts of Chemical Research Jun 2002All-atom molecular dynamics simulations of proteins in solvent are now able to realistically map the protein-unfolding pathway. The agreement with experiments probing... (Review)
Review
All-atom molecular dynamics simulations of proteins in solvent are now able to realistically map the protein-unfolding pathway. The agreement with experiments probing both folding and unfolding suggests that these simulated unfolding events also shed light on folding. The simulations have produced detailed models of protein folding transition, intermediate, and denatured states that are in both qualitative and quantitative agreement with experiment. The various studies presented here highlight how such simulations both complement and extend experiment.
Topics: Computer Simulation; Motion; Protein Conformation; Protein Denaturation; Protein Folding; Proteins
PubMed: 12069627
DOI: 10.1021/ar0100834 -
Small (Weinheim An Der Bergstrasse,... Sep 2017Spatiotemporal control of protein structure and activity in biological systems has important and broad implications in biomedical sciences as evidenced by recent...
Spatiotemporal control of protein structure and activity in biological systems has important and broad implications in biomedical sciences as evidenced by recent advances in optogenetic approaches. Here, this study demonstrates that nanosecond pulsed laser heating of gold nanoparticles (GNP) leads to an ultrahigh and ultrashort temperature increase, coined as "molecular hyperthermia", which causes selective unfolding and inactivation of proteins adjacent to the GNP. Protein inactivation is highly dependent on both laser pulse energy and GNP size, and has a well-defined impact zone in the nanometer scale. It is anticipated that the fine control over protein structure and function enabled by this discovery will be highly enabling within a number of arenas, from probing the biophysics of protein folding/unfolding to the nanoscopic manipulation of biological systems via an optical trigger, to developing novel therapeutics for disease treatment without genetic modification.
Topics: Gold; Hot Temperature; Metal Nanoparticles; Protein Unfolding; Proteins; Time Factors
PubMed: 28696524
DOI: 10.1002/smll.201700841 -
ACS Nano Jul 2021Hierarchical assemblies of proteins exhibit a wide-range of material properties that are exploited both in nature and by artificially by humankind. However, little is...
Hierarchical assemblies of proteins exhibit a wide-range of material properties that are exploited both in nature and by artificially by humankind. However, little is understood about the importance of protein unfolding on the network assembly, severely limiting opportunities to utilize this nanoscale transition in the development of biomimetic and bioinspired materials. Here we control the force lability of a single protein building block, bovine serum albumin (BSA), and demonstrate that protein unfolding plays a critical role in defining the architecture and mechanics of a photochemically cross-linked native protein network. The internal nanoscale structure of BSA contains "molecular reinforcement" in the form of 17 covalent disulphide "nanostaples", preventing force-induced unfolding. Upon addition of reducing agents, these nanostaples are broken rendering the protein force labile. Employing a combination of circular dichroism (CD) spectroscopy, small-angle scattering (SAS), rheology, and modeling, we show that stapled protein forms reasonably homogeneous networks of cross-linked fractal-like clusters connected by an intercluster region of folded protein. Conversely, protein unfolding results in more heterogeneous networks of denser fractal-like clusters connected by an intercluster region populated by unfolded protein. In addition, gelation-induced protein unfolding and cross-linking in the intercluster region changes the hydrogel mechanics, as measured by a 3-fold enhancement of the storage modulus, an increase in both the loss ratio and energy dissipation, and markedly different relaxation behavior. By controlling the protein's ability to unfold through nanoscale (un)stapling, we demonstrate the importance of unfolding in defining both network architecture and mechanics, providing insight into fundamental hierarchical mechanics and a route to tune biomaterials for future applications.
Topics: Hydrogels; Protein Unfolding; Biocompatible Materials; Serum Albumin, Bovine; Rheology
PubMed: 34214394
DOI: 10.1021/acsnano.1c00353 -
Biochemical and Biophysical Research... May 2020CPAP is a centriolar protein and its C-terminal domain, G-box or TCP, has a very unique structure that comprises a single-layer β-sheet without hydrophobic core...
CPAP is a centriolar protein and its C-terminal domain, G-box or TCP, has a very unique structure that comprises a single-layer β-sheet without hydrophobic core packing. Here we characterized its biophysical properties, including its stability against chemical denaturation. Interestingly, upon urea-induced equilibrium unfolding, the CPAP G-box showed cooperative unfolding behavior that is the hallmark of globular proteins. We analyzed the m-value, a measure of the cooperative transition, from the urea-induced unfolding and found that the estimated m-value from surface burial upon folding is consistent with the experimental value, supporting the two-state unfolding. Next, we constructed deletion mutants of the terminal β-strands and found that the mutants showed reduced stability. The unique structure and characteristics of CPAP G-box provides an interesting opportunity to observe how the core-less flat β-sheet protein can be folded in solution.
Topics: Animals; Microtubule-Associated Proteins; Protein Denaturation; Protein Domains; Protein Stability; Protein Structure, Secondary; Protein Unfolding; Sequence Deletion; Zebrafish; Zebrafish Proteins
PubMed: 32197835
DOI: 10.1016/j.bbrc.2020.03.056 -
Molecular Pharmaceutics Jul 2020Determining the temperature at which the thermal unfolding of a protein starts becoming irreversible is relevant for many areas of protein research. Until now, published...
Determining the temperature at which the thermal unfolding of a protein starts becoming irreversible is relevant for many areas of protein research. Until now, published methods cannot determine, within a reasonable time frame and with moderate sample consumption, the exposure temperature that starts causing irreversible protein unfolding. We present modulated scanning fluorimetry (MSF) and share a software (MSF Analyzer), which can be used to derive nonreversibility curves of thermal protein unfolding from a series of incremental temperature cycles performed on only 10 μL samples, consuming as low as a few micrograms of protein. Further processing of the data can yield the onset temperature that starts causing nonreversible protein unfolding. The MSF method is based on the hardware of the already existing nanoDSF technology and can be applied to dozens of samples simultaneously. Here, we use MSF to study how solution pH affects the reversibility of thermal protein unfolding of several model proteins to show that the nonreversibility onset temperature () is a unique biophysical parameter, providing orthogonal information from thermal protein denaturation data and insights into the validity of thermal unfolding analysis in the context of equilibrium thermodynamics. We also show that MSF can be used to study enzyme stability after exposure to high temperatures. Besides, we demonstrate that protein thermal unfolding and nonreversibility can be affected in different ways upon modifications like PEG-ylation or labeling with fluorescent dyes. Finally, we show that MSF can be used to study the effect of various protein interactions on thermal protein unfolding reversibility. With the diverse examples in this work, we reveal how MSF can provide orthogonal information from thermal denaturation experiments that can bring benefits to various areas of protein research. The MSF Analyzer software is available at https://github.com/CoriolisPharmaResearch/MSFAnalyser.
Topics: Calorimetry, Differential Scanning; Fluorescent Dyes; Fluorometry; Hot Temperature; Hydrogen-Ion Concentration; Kinetics; Muramidase; Ovalbumin; Polyethylene Glycols; Protein Denaturation; Protein Folding; Protein Unfolding; Proteins; Software; Thermodynamics; Trastuzumab; Ubiquitin
PubMed: 32401526
DOI: 10.1021/acs.molpharmaceut.0c00330 -
Angewandte Chemie (International Ed. in... Sep 2014Aptides, a novel class of high-affinity peptides, recognize diverse molecular targets with high affinity and specificity. The solution structure of the aptide APT...
Aptides, a novel class of high-affinity peptides, recognize diverse molecular targets with high affinity and specificity. The solution structure of the aptide APT specifically bound to fibronectin extradomain B (EDB), which represents an unusual protein-protein interaction that involves coupled unfolding and binding, is reported. APT binding is accompanied by unfolding of the C-terminal β strand of EDB, thereby permitting APT to interact with the freshly exposed hydrophobic interior surfaces of EDB. The β-hairpin scaffold of APT drives the interaction by a β-strand displacement mechanism, such that an intramolecular β sheet is replaced by an intermolecular β sheet. The unfolding of EDB perturbs the tight domain association between EDB and FN8 of fibronectin, thus highlighting its potential use as a scaffold that switches between stretched and bent conformations.
Topics: Fibronectins; Magnetic Resonance Spectroscopy; Models, Molecular; Protein Binding; Protein Interaction Domains and Motifs; Protein Unfolding
PubMed: 24985319
DOI: 10.1002/anie.201404750 -
The Journal of Physical Chemistry. B Jun 2020Thermal protein unfolding resembles a global (two-state) phase transition. At the local scale, protein unfolding is, however, heterogeneous and probe dependent. Here, we...
Thermal protein unfolding resembles a global (two-state) phase transition. At the local scale, protein unfolding is, however, heterogeneous and probe dependent. Here, we consider local order parameters defined by the local curvature and torsion of the protein main chain. Because chemical shifts (CS's) measured by NMR spectroscopy are extremely sensitive to the local atomic environment, CS has served as a local probe of thermal unfolding of proteins by varying the position of the atomic isotope along the amino acid sequence. The variation of the CS of each C atom along the sequence as a function of the temperature defines a local heat-induced denaturation curve. We demonstrate that these local heat-induced denaturation curves mirror the local protein nativeness defined by the free energy landscape of the local curvature and torsion of the protein main chain described by the CC virtual bonds. Comparison between molecular dynamics simulations and CS data of the gpW protein demonstrates that some local native states defined by the local curvature and torsion of the main chain, mainly located in secondary structures, are coupled to each other whereas others, mainly located in flexible protein segments, are not. Consequently, CS's of some residues are faithful reporters of global protein unfolding, with heat-induced denaturation curves similar to the average global one, whereas other residues remain silent about the protein unfolded state. For the latter, the local deformation of the protein main chain, characterized by its local curvature and torsion, is not cooperatively coupled to global unfolding.
Topics: Amino Acid Sequence; Protein Conformation; Protein Denaturation; Protein Folding; Protein Structure, Secondary; Protein Unfolding; Thermodynamics
PubMed: 32392067
DOI: 10.1021/acs.jpcb.0c01230