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International Journal of Molecular... Mar 2023We review the key steps leading to an improved analysis of thermal protein unfolding. Thermal unfolding is a dynamic cooperative process with many short-lived... (Review)
Review
We review the key steps leading to an improved analysis of thermal protein unfolding. Thermal unfolding is a dynamic cooperative process with many short-lived intermediates. Protein unfolding has been measured by various spectroscopic techniques that reveal structural changes, and by differential scanning calorimetry (DSC) that provides the heat capacity change C(T). The corresponding temperature profiles of enthalpy ΔH(T), entropy ΔS(T), and free energy ΔG(T) have thus far been evaluated using a chemical equilibrium two-state model. Taking a different approach, we demonstrated that the temperature profiles of enthalpy ΔH(T), entropy ΔS(T), and free energy ΔG(T) can be obtained directly by a numerical integration of the heat capacity profile C(T). DSC thus offers the unique possibility to assess these parameters without resorting to a model. These experimental parameters now allow us to examine the predictions of different unfolding models. The standard two-state model fits the experimental heat capacity peak quite well. However, neither the enthalpy nor entropy profiles (predicted to be almost linear) are congruent with the measured sigmoidal temperature profiles, nor is the parabolic free energy profile congruent with the experimentally observed trapezoidal temperature profile. We introduce three new models, an empirical two-state model, a statistical-mechanical two-state model and a cooperative statistical-mechanical multistate model. The empirical model partially corrects for the deficits of the standard model. However, only the two statistical-mechanical models are thermodynamically consistent. The two-state models yield good fits for the enthalpy, entropy and free energy of unfolding of small proteins. The cooperative statistical-mechanical multistate model yields perfect fits, even for the unfolding of large proteins such as antibodies.
Topics: Protein Denaturation; Thermodynamics; Protein Unfolding; Entropy; Proteins; Calorimetry, Differential Scanning; Protein Folding
PubMed: 36982534
DOI: 10.3390/ijms24065457 -
Journal of Molecular Biology Apr 2019Protein folding/unfolding is a complicated process that defies high-resolution characterization by experimental methods. As an alternative, atomistic molecular dynamics... (Review)
Review
Protein folding/unfolding is a complicated process that defies high-resolution characterization by experimental methods. As an alternative, atomistic molecular dynamics simulations are now routinely employed to elucidate and magnify the accompanying conformational changes and the role of solvent in the folding process. However, the level of detail necessary to map the process at high spatial-temporal resolution provides an overwhelming amount of data. As more and better tools are developed for analysis of these large data sets and validation of the simulations, one is still left with the problem of visualizing the results in ways that provide insight into the folding/unfolding process. While viewing and interrogating static crystal structures has become commonplace, more and different approaches are required for dynamic, interconverting, unfolding, and refolding proteins. Here we review a variety of approaches, ranging from straightforward to complex and unintuitive for multiscale analysis and visualization of protein folding and unfolding.
Topics: Animals; Humans; Molecular Dynamics Simulation; Protein Conformation; Protein Folding; Protein Unfolding; Proteins
PubMed: 30840846
DOI: 10.1016/j.jmb.2019.02.026 -
Annual Review of Physiology Feb 2018AAA+ proteolytic machines use energy from ATP hydrolysis to degrade damaged, misfolded, or unneeded proteins. Protein degradation occurs within a barrel-shaped... (Review)
Review
AAA+ proteolytic machines use energy from ATP hydrolysis to degrade damaged, misfolded, or unneeded proteins. Protein degradation occurs within a barrel-shaped self-compartmentalized peptidase. Before protein substrates can enter this peptidase, they must be unfolded and then translocated through the axial pore of an AAA+ ring hexamer. An unstructured region of the protein substrate is initially engaged in the axial pore, and conformational changes in the ring, powered by ATP hydrolysis, generate a mechanical force that pulls on and denatures the substrate. The same conformational changes in the hexameric ring then mediate mechanical translocation of the unfolded polypeptide into the peptidase chamber. For the bacterial ClpXP and ClpAP AAA+ proteases, the mechanical activities of protein unfolding and translocation have been directly visualized by single-molecule optical trapping. These studies in combination with structural and biochemical experiments illuminate many principles that underlie this universal mechanism of ATP-fueled protein unfolding and subsequent destruction.
Topics: Adenosine Triphosphate; Animals; Humans; Protein Unfolding; Proteolysis
PubMed: 29433415
DOI: 10.1146/annurev-physiol-021317-121303 -
Advances in Experimental Medicine and... 2021Single molecule force spectroscopy (SMFS) has emerged since the past few years as a prominent set of techniques, within the broader field of atomic force microscopy... (Review)
Review
Single molecule force spectroscopy (SMFS) has emerged since the past few years as a prominent set of techniques, within the broader field of atomic force microscopy (AFM), for the study of interactions and binding forces of individual protein molecules. Since force spectroscopy measures the behavior of a molecule when stretching or torsional mechanical force is applied, it can be an excellent tool in the hands of researchers who study protein folding and misfolding mechanisms, by reverse engineering the forced unfolding. Such studies could be of crucial importance in the field of protein-related diseases. In this review we wish to provide a glimpse at SMFS concept and recent advances, paired with the protein misfolding issue in neurodegenerative diseases.
Topics: Microscopy, Atomic Force; Protein Domains; Protein Folding; Protein Unfolding; Single Molecule Imaging; Spectrum Analysis
PubMed: 35023106
DOI: 10.1007/978-3-030-78787-5_24 -
Protein and Peptide Letters Mar 2014In this mini-review we introduce and discuss a new method, at single molecule level, to study the protein folding and protein stability, with a nanopore coupled to an... (Review)
Review
In this mini-review we introduce and discuss a new method, at single molecule level, to study the protein folding and protein stability, with a nanopore coupled to an electric detection. Proteins unfolded or partially folded passing through one channel submitted to an electric field, in the presence of salt solution, induce different detectable blockades of ionic current. Their duration depends on protein conformation. For different studies proteins through nanopores, completely unfolded proteins induce only short current blockades. Their frequency increases as the concentration of denaturing agent or temperature increases, following a sigmoidal denaturation curve. The geometry or the net charge of the nanopores does not alter the unfolding transition, sigmoidal unfolding curve and half denaturing concentration or half temperature denaturation. A destabilized protein induces a shift of the unfolding curve towards the lower values of the denaturant agent compared to the wild type protein.Partially folded proteins exhibit very long blockades in nanopores. The blockade duration decreases when the concentration of denaturing agent increases. The variation of these blockades could be associated to a possible glassy behaviour.
Topics: Animals; Biophysics; Humans; Models, Molecular; Nanopores; Protein Conformation; Protein Denaturation; Protein Stability; Protein Unfolding; Proteins
PubMed: 24370253
DOI: 10.2174/09298665113209990080 -
The FEBS Journal Mar 2021Unfolding and refolding of multidomain proteins under force have yet to be recognized as a major mechanism of function for proteins in vivo. In this review, we discuss... (Review)
Review
Unfolding and refolding of multidomain proteins under force have yet to be recognized as a major mechanism of function for proteins in vivo. In this review, we discuss the inherent properties of multidomain proteins under a force vector from a structural and functional perspective. We then characterize three main systems where multidomain proteins could play major roles through mechanical unfolding: muscular contraction, cellular mechanotransduction, and bacterial adhesion. We analyze how key multidomain proteins for each system can produce a gain-of-function from the perspective of a fine-tuned quantized response, a molecular battery, delivery of mechanical work through refolding, elasticity tuning, protection and exposure of cryptic sites, and binding-induced mechanical changes. Understanding how mechanical unfolding and refolding affect function will have important implications in designing mechano-active drugs against conditions such as muscular dystrophy, cancer, or novel antibiotics.
Topics: Elasticity; Mechanotransduction, Cellular; Models, Molecular; Protein Binding; Protein Domains; Protein Folding; Protein Unfolding; Proteins; Stress, Mechanical; Thermodynamics
PubMed: 32761965
DOI: 10.1111/febs.15508 -
Chemphyschem : a European Journal of... Jan 2022Approximating protein unfolding by an all-or-none cooperative event is a convenient assumption that can provide precious global information on protein stability. It is... (Review)
Review
Approximating protein unfolding by an all-or-none cooperative event is a convenient assumption that can provide precious global information on protein stability. It is however quickly emerging that the scenario is far more complex and that global denaturation curves often hide a rich heterogeneity of states that are largely probe dependent. In this review, we revisit the importance of gaining site-specific information on the unfolding process. We focus on nuclear magnetic resonance, as this is the main technique able to provide site-specific information. We review historical and most modern approaches that have allowed an appreciable advancement of the field of protein folding. We also demonstrate how unfolding is a reporter dependent event, suggesting the outmost importance of selecting the reporter carefully.
Topics: Circular Dichroism; Onions; Protein Denaturation; Protein Folding; Protein Unfolding; Thermodynamics
PubMed: 34549492
DOI: 10.1002/cphc.202100520 -
Methods in Molecular Biology (Clifton,... 2023Maintaining protein homeostasis (proteostasis) is vital to cellular and organismal health. How the Golgi apparatus, the central protein maturation and sorting station in...
Maintaining protein homeostasis (proteostasis) is vital to cellular and organismal health. How the Golgi apparatus, the central protein maturation and sorting station in the cell, manages misfolded proteins to maintain proteostasis is still poorly understood. Here we present a strategy for targeted protein unfolding at the Golgi that enables studying Golgi-related protein quality control and stress-signaling pathways. Targeted protein unfolding is induced by small molecule-based chemical biology approaches-hydrophobic tagging and the use of a destabilization domain. Imaging studies allow visualizing quality control (QC) phenotypes, such as the formation of QC carriers and Golgi-to-endoplasmic reticulum trafficking, and correlating these phenotypes with other trafficking processes.
Topics: Golgi Apparatus; Endoplasmic Reticulum; Protein Transport; Protein Unfolding
PubMed: 36512243
DOI: 10.1007/978-1-0716-2639-9_39 -
Journal of Biomolecular NMR Apr 2022NMR-spectroscopy has certain unique advantages for recording unfolding transitions of proteins compared e.g. to optical methods. It enables per-residue monitoring and...
NMR-spectroscopy has certain unique advantages for recording unfolding transitions of proteins compared e.g. to optical methods. It enables per-residue monitoring and separate detection of the folded and unfolded state as well as possible equilibrium intermediates. This allows a detailed view on the state and cooperativity of folding of the protein of interest and the correct interpretation of subsequent experiments. Here we summarize in detail practical and theoretical aspects of such experiments. Certain pitfalls can be avoided, and meaningful simplification can be made during the analysis. Especially a good understanding of the NMR exchange regime and relaxation properties of the system of interest is beneficial. We show by a global analysis of signals of the folded and unfolded state of GB1 how accurate values of unfolding can be extracted and what limits different NMR detection and unfolding methods. E.g. commonly used exchangeable amides can lead to a systematic under determination of the thermodynamic protein stability. We give several perspectives of how to deal with more complex proteins and how the knowledge about protein stability at residue resolution helps to understand protein properties under crowding conditions, during phase separation and under high pressure.
Topics: Magnetic Resonance Spectroscopy; Nuclear Magnetic Resonance, Biomolecular; Protein Denaturation; Protein Folding; Protein Unfolding; Proteins; Thermodynamics
PubMed: 34984658
DOI: 10.1007/s10858-021-00389-3 -
Current Opinion in Structural Biology Feb 2017Extracellular matrix fibrils of fibronectin (FN) are highly elastic, and are typically stretched three to four times their relaxed length. The mechanism of stretching... (Review)
Review
Extracellular matrix fibrils of fibronectin (FN) are highly elastic, and are typically stretched three to four times their relaxed length. The mechanism of stretching has been controversial, in particular whether it involves tension-induced unfolding of FNIII domains. Recent studies have found that ∼5pN is the threshold isometric force for unfolding various protein domains. FNIII domains should therefore not be unfolded until the tension approaches 5pN. Integrins have been reported to generate forces ranging from 1 to >50pN, but I argue that studies reporting 1-2pN are the most convincing. This is not enough to unfold FNIII domains. Even if domains were unfolded, 2pN would only extend the worm-like-chain to about twice the length of the folded domain. Overall I conclude that stretching FN matrix fibrils involves primarily the compact to extended conformational change of FN dimers, with minimal contribution from unfolding FNIII domains.
Topics: Fibronectins; Integrins; Mechanical Phenomena; Protein Domains; Protein Refolding; Protein Unfolding
PubMed: 28038331
DOI: 10.1016/j.sbi.2016.12.002