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International Journal of Molecular... Nov 2022Many proteins form amyloid fibrils only under conditions when the probability of transition from a native (structured, densely packed) to an intermediate (labile,...
Many proteins form amyloid fibrils only under conditions when the probability of transition from a native (structured, densely packed) to an intermediate (labile, destabilized) state is increased. It implies the assumption that some structural intermediates are more convenient for amyloid formation than the others. Hence, if a mutation affects the protein folding pathway, one should expect that this mutation could affect the rate of amyloid formation as well. In the current work, we have compared the effects of amino acid substitutions of bovine carbonic anhydrase II on its unfolding pathway and on its ability to form amyloids at acidic pH and an elevated temperature. Wild-type protein and four mutant forms (L78A, L139A, I208A, and M239A) were studied. We analyzed the change of the protein unfolding pathway by the time-resolved fluorescence technique and the process of amyloid formation by thioflavin T fluorescence assay and electron microscopy. It was revealed that I208A substitution accelerates amyloid formation and affects the structure of the late (molten globule-like)-intermediate state of carbonic anhydrase, whereas the other mutations slow down the growth of amyloids and have either no effect on the unfolding pathway (L78A, L139A) or alter the conformational states arising at the early unfolding stage (M239A).
Topics: Cattle; Animals; Carbonic Anhydrase II; Protein Folding; Amyloid; Carbonic Anhydrases; Amyloidogenic Proteins; Protein Conformation; Protein Denaturation; Circular Dichroism
PubMed: 36498970
DOI: 10.3390/ijms232314645 -
The Journal of Physical Chemistry. B Oct 2019Cells of the vast majority of organisms are subject to temperature, pressure, pH, ionic strength, and other stresses. We discuss these effects in the light of protein... (Review)
Review
Cells of the vast majority of organisms are subject to temperature, pressure, pH, ionic strength, and other stresses. We discuss these effects in the light of protein folding and protein interactions , in complex environments, in cells, and . Protein phase diagrams provide a way of organizing different structural ensembles that occur under stress and how one can move among ensembles. Experiments that perturb biomolecules or in cells by stressing them have revealed much about the underlying forces that are competing to control protein stability, folding, and function. Two phenomena that emerge and serve to broadly classify effects of the cellular environment are crowding (mainly due to repulsive forces) and sticking (mainly due to attractive forces). The interior of cells is closely balanced between these emergent effects, and stress can tip the balance one way or the other. The free energy scale involved is small but significant on the scale of the "on/off switches" that control signaling in cells or of protein-protein association with a favorable function such as increased enzyme processivity. Quantitative tools from biophysical chemistry will play an important role in elucidating the world of crowding and sticking under stress.
Topics: Animals; Humans; Protein Binding; Protein Folding; Proteins; Stress, Physiological
PubMed: 31386813
DOI: 10.1021/acs.jpcb.9b05467 -
Cell Stress & Chaperones Jun 1996Protein folding inside the cell involves the participation of accessory components known as molecular chaperones. In addition to their active participation in the... (Review)
Review
Protein folding inside the cell involves the participation of accessory components known as molecular chaperones. In addition to their active participation in the folding process, molecular chaperones serve as a type of 'quality control system', recognizing, retaining and targeting misfolded proteins for their eventual degradation. It is now known that a number of human diseases arise as a consequence of specific point mutations or deletions within genes encoding essential proteins. In many cases these mutations/deletions are not so severe as to totally destroy the biological activity of the particular gene product. Rather, the mutations often result in only subtle folding abnormalities which lead to the newly synthesized protein being retained at the endoplasmic reticulum by the actions of the cellular quality control system. In this short review article we discuss our recent studies showing that the protein folding defect associated with the most common mutation in patients with cystic fibrosis can be overcome by a novel strategy. As shown in the paper by Brown et al in this issue (Brown et al 1996), a number of different low molecular weight compounds, all known to stabilize proteins in their native conformation, are effective in rescuing the processing defect of the mutant cystic fibrosis transmembrane conductance regulator protein. We then discuss how these same compounds, which we now call chemical chaperones, also may prove to be effective in correcting a number of other protein folding abnormalities which constitute the underlying basis of a large number of different human diseases.
Topics: Humans; Molecular Chaperones; Protein Folding
PubMed: 9222596
DOI: 10.1379/1466-1268(1996)001<0109:iomacc>2.3.co;2 -
Journal of Molecular Biology Oct 2023The study of protein folding plays a crucial role in improving our understanding of protein function and of the relationship between genetics and phenotypes. In...
The study of protein folding plays a crucial role in improving our understanding of protein function and of the relationship between genetics and phenotypes. In particular, understanding the thermodynamics and kinetics of the folding process is important for uncovering the mechanisms behind human disorders caused by protein misfolding. To address this issue, it is essential to collect and curate experimental kinetic and thermodynamic data on protein folding. K-Pro is a new database designed for collecting and storing experimental kinetic data on monomeric proteins, with a two-state folding mechanism. With 1,529 records from 62 proteins corresponding to 65 structures, K-Pro contains various kinetic parameters such as the logarithm of the folding and unfolding rates, Tanford's β and the ϕ values. When available, the database also includes thermodynamic parameters associated with the kinetic data. K-Pro features a user-friendly interface that allows browsing and downloading kinetic data of interest. The graphical interface provides a visual representation of the protein and mutants, and it is cross-linked to key databases such as PDB, UniProt, and PubMed. K-Pro is open and freely accessible through https://folding.biofold.org/k-pro and supports the latest versions of popular browsers.
Topics: Humans; Databases, Protein; Kinetics; Protein Denaturation; Protein Folding; Proteins; Thermodynamics; Mutant Proteins
PubMed: 37625584
DOI: 10.1016/j.jmb.2023.168245 -
Protein Science : a Publication of the... Aug 2008This review compares the folding behavior of proteins and RNAs. Topics covered include the role of topology in the determination of folding rates, major folding events... (Review)
Review
This review compares the folding behavior of proteins and RNAs. Topics covered include the role of topology in the determination of folding rates, major folding events including collapse, properties of denatured states, pathway heterogeneity, and the influence of the mode of initiation on the folding pathway.
Topics: Animals; Humans; Models, Molecular; Nucleic Acid Conformation; Nucleic Acid Denaturation; Protein Denaturation; Protein Folding; Protein Structure, Secondary; Proteins; RNA
PubMed: 18502978
DOI: 10.1110/ps.036319.108 -
The Journal of Physical Chemistry. B Apr 2023Protein stability is important in many areas of life sciences. Thermal protein unfolding is investigated extensively with various spectroscopic techniques. The...
Protein stability is important in many areas of life sciences. Thermal protein unfolding is investigated extensively with various spectroscopic techniques. The extraction of thermodynamic properties from these measurements requires the application of models. Differential scanning calorimetry (DSC) is less common, but is unique as it measures directly a thermodynamic property, that is, the heat capacity (). The analysis of () is usually performed with the chemical equilibrium two-state model. This is not necessary and leads to incorrect thermodynamic consequences. Here we demonstrate a straightforward model-independent evaluation of heat capacity experiments in terms of protein unfolding enthalpy Δ(), entropy Δ(), and free energy Δ()). This now allows the comparison of the experimental thermodynamic data with the predictions of different models. We critically examined the standard chemical equilibrium two-state model, which predicts a positive free energy for the native protein, and diverges distinctly from the experimental temperature profiles. We propose two new models which are equally applicable to spectroscopy and calorimetry. The Θ()-weighted chemical equilibrium model and the statistical-mechanical two-state model provide excellent fits of the experimental data. They predict sigmoidal temperature profiles for enthalpy and entropy, and a trapezoidal temperature profile for the free energy. This is illustrated with experimental examples for heat and cold denaturation of lysozyme and β-lactoglobulin. We then show that the free energy is not a good criterion to judge protein stability. More useful parameters are discussed, including protein cooperativity. The new parameters are embedded in a well-defined thermodynamic context and are amenable to molecular dynamics calculations.
Topics: Hot Temperature; Protein Denaturation; Proteins; Thermodynamics; Cold Temperature; Protein Unfolding; Calorimetry, Differential Scanning; Protein Folding
PubMed: 37040567
DOI: 10.1021/acs.jpcb.3c00882 -
Biophysical Journal Dec 2016A complete description of the pathways and mechanisms of protein folding requires a detailed structural and energetic characterization of the conformational ensemble...
A complete description of the pathways and mechanisms of protein folding requires a detailed structural and energetic characterization of the conformational ensemble along the entire folding reaction coordinate. Simulations can provide this level of insight for small proteins. In contrast, with the exception of hydrogen exchange, which does not monitor folding directly, experimental studies of protein folding have not yielded such structural and energetic detail. NMR can provide residue specific atomic level structural information, but its implementation in protein folding studies using chemical or temperature perturbation is problematic. Here we present a highly detailed structural and energetic map of the entire folding landscape of the leucine-rich repeat protein, pp32 (Anp32), obtained by combining pressure-dependent site-specific H-N HSQC data with coarse-grained molecular dynamics simulations. The results obtained using this equilibrium approach demonstrate that the main barrier to folding of pp32 is quite broad and lies near the unfolded state, with structure apparent only in the C-terminal region. Significant deviation from two-state unfolding under pressure reveals an intermediate on the folded side of the main barrier in which the N-terminal region is disordered. A nonlinear temperature dependence of the population of this intermediate suggests a large heat capacity change associated with its formation. The combination of pressure, which favors the population of folding intermediates relative to chemical denaturants; NMR, which allows their observation; and constrained structure-based simulations yield unparalleled insight into protein folding mechanisms.
Topics: Amino Acid Sequence; Intracellular Signaling Peptides and Proteins; Models, Molecular; Pressure; Protein Domains; Protein Folding; Protein Unfolding; Thermodynamics
PubMed: 27926838
DOI: 10.1016/j.bpj.2016.08.027 -
Analytical Chemistry Jan 2015Theta-glass emitters are used to rapidly mix two solutions to induce either protein folding or unfolding during nanoelectrospray (nanoESI). Mixing acid-denatured...
Theta-glass emitters are used to rapidly mix two solutions to induce either protein folding or unfolding during nanoelectrospray (nanoESI). Mixing acid-denatured myoglobin with an aqueous ammonium acetate solution to increase solution pH results in protein folding during nanoESI. A reaction time and upper limit to the droplet lifetime of 9 ± 2 μs is obtained from the relative abundance of the folded conformer in these rapid mixing experiments compared to that obtained from solutions at equilibrium and a folding time constant of 7 μs. Heme reincorporation does not occur, consistent with the short droplet lifetime and the much longer time constant for this process. Similar mixing experiments with acid-denatured cytochrome c and the resulting folding during nanoESI indicate a reaction time of between 7 and 25 μs depending on the solution composition. The extent of unfolding of holo-myoglobin upon rapid mixing with theta-glass emitters is less than that reported previously ( Fisher et al. Anal. Chem. 2014 , 86 , 4581 - 4588 ), a result that is attributed to the much smaller, ∼1.5 μm, average o.d. tips used here. These results indicate that the time frame during which protein folding or unfolding can occur during nanoESI depends both on the initial droplet size, which can be varied by changing the emitter tip diameter, and on the solution composition. This study demonstrates that protein folding or unfolding processes that occur on the ∼10 μs time scale can be readily investigated using rapid mixing with theta-glass emitters combined with mass spectrometry.
Topics: Animals; Cytochromes c; Heme; Horses; Kinetics; Myoglobin; Protein Denaturation; Protein Folding; Protein Unfolding; Spectrometry, Mass, Electrospray Ionization
PubMed: 25525976
DOI: 10.1021/ac503981c -
ACS Nano Jul 2017Single-molecule studies of protein folding hold keys to unveiling protein folding pathways and elusive intermediate folding states-attractive pharmaceutical targets....
Single-molecule studies of protein folding hold keys to unveiling protein folding pathways and elusive intermediate folding states-attractive pharmaceutical targets. Although conventional single-molecule approaches can detect folding intermediates, they presently lack throughput and require elaborate labeling. Here, we theoretically show that measurements of ionic current through a nanopore containing a protein can report on the protein's folding state. Our all-atom molecular dynamics (MD) simulations show that the unfolding of a protein lowers the nanopore ionic current, an effect that originates from the reduction of ion mobility in proximity to a protein. Using a theoretical model, we show that the average change in ionic current produced by a folding-unfolding transition is detectable despite the orientational and conformational heterogeneity of the folded and unfolded states. By analyzing millisecond-long all-atom MD simulations of multiple protein transitions, we show that a nanopore ionic current recording can detect folding-unfolding transitions in real time and report on the structure of folding intermediates.
Topics: Databases, Protein; Ion Transport; Molecular Dynamics Simulation; Nanopores; Protein Conformation; Protein Folding; Protein Unfolding; Proteins
PubMed: 28693322
DOI: 10.1021/acsnano.7b02718 -
Proceedings of the National Academy of... Oct 2013Protein folding has been extensively studied, but many questions remain regarding the mechanism. Characterizing early unstable intermediates and the high-free-energy...
Protein folding has been extensively studied, but many questions remain regarding the mechanism. Characterizing early unstable intermediates and the high-free-energy transition state (TS) will help answer some of these. Here, we use effects of denaturants (urea, guanidinium chloride) and temperature on folding and unfolding rate constants and the overall equilibrium constant as probes of surface area changes in protein folding. We interpret denaturant kinetic m-values and activation heat capacity changes for 13 proteins to determine amounts of hydrocarbon and amide surface buried in folding to and from TS, and for complete folding. Predicted accessible surface area changes for complete folding agree in most cases with structurally determined values. We find that TS is advanced (50-90% of overall surface burial) and that the surface buried is disproportionately amide, demonstrating extensive formation of secondary structure in early intermediates. Models of possible pre-TS intermediates with all elements of the native secondary structure, created for several of these proteins, bury less amide and hydrocarbon surface than predicted for TS. Therefore, we propose that TS generally has both the native secondary structure and sufficient organization of other regions of the backbone to nucleate subsequent (post-TS) formation of tertiary interactions. The approach developed here provides proof of concept for the use of denaturants and other solutes as probes of amount and composition of the surface buried in coupled folding and other large conformational changes in TS and intermediates in protein processes.
Topics: Models, Chemical; Protein Denaturation; Protein Folding; Proteins
PubMed: 24043778
DOI: 10.1073/pnas.1311948110