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Microbial Cell Factories Mar 2015Formation of inclusion bodies in bacterial hosts poses a major challenge for large scale recovery of bioactive proteins. The process of obtaining bioactive protein from... (Review)
Review
Formation of inclusion bodies in bacterial hosts poses a major challenge for large scale recovery of bioactive proteins. The process of obtaining bioactive protein from inclusion bodies is labor intensive and the yields of recombinant protein are often low. Here we review the developments in the field that are targeted at improving the yield, as well as quality of the recombinant protein by optimizing the individual steps of the process, especially solubilization of the inclusion bodies and refolding of the solubilized protein. Mild solubilization methods have been discussed which are based on the understanding of the fact that protein molecules in inclusion body aggregates have native-like structure. These methods solubilize the inclusion body aggregates while preserving the native-like protein structure. Subsequent protein refolding and purification results in high recovery of bioactive protein. Other parameters which influence the overall recovery of bioactive protein from inclusion bodies have also been discussed. A schematic model describing the utility of mild solubilization methods for high throughput recovery of bioactive protein has also been presented.
Topics: Escherichia coli; Inclusion Bodies; Models, Molecular; Protein Denaturation; Protein Folding; Protein Refolding; Protein Unfolding; Recombinant Proteins; Solubility
PubMed: 25889252
DOI: 10.1186/s12934-015-0222-8 -
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 -
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 -
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 -
Chembiochem : a European Journal of... Aug 2023This review aims to analyse the role of solution nuclear magnetic resonance spectroscopy in pressure-induced in vitro studies of protein unfolding. Although this... (Review)
Review
This review aims to analyse the role of solution nuclear magnetic resonance spectroscopy in pressure-induced in vitro studies of protein unfolding. Although this transition has been neglected for many years because of technical difficulties, it provides important information about the forces that keep protein structure together. We first analyse what pressure unfolding is, then provide a critical overview of how NMR spectroscopy has contributed to the field and evaluate the observables used in these studies. Finally, we discuss the commonalities and differences between pressure-, cold- and heat-induced unfolding. We conclude that, despite specific peculiarities, in both cold and pressure denaturation the important contribution of the state of hydration of nonpolar side chains is a major factor that determines the pressure dependence of the conformational stability of proteins.
Topics: Protein Denaturation; Proteins; Magnetic Resonance Spectroscopy; Protein Unfolding; Protein Conformation; Thermodynamics; Protein Folding; Cold Temperature
PubMed: 37154795
DOI: 10.1002/cbic.202300164 -
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 -
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 -
Current Opinion in Structural Biology Feb 2016Theory and simulations predict that the structural concert of protein folding reactions is relatively low. Experimentally, folding cooperativity has been difficult to... (Review)
Review
Theory and simulations predict that the structural concert of protein folding reactions is relatively low. Experimentally, folding cooperativity has been difficult to study, but in recent years we have witnessed major advances. New analytical procedures in terms of conformational ensembles rather than discrete states, experimental techniques with improved time, structural, or single-molecule resolution, and combined thermodynamic and kinetic analysis of fast folding have contributed to demonstrate a general scenario of limited cooperativity in folding. Gradual structural disorder is already apparent on the unfolded and native states of slow, two-state folding proteins, and it greatly increases in magnitude for fast folding domains. These results demonstrate a direct link between how fast a single-domain protein folds and unfolds, and how cooperative (or structurally diverse) is its equilibrium unfolding process. Reducing cooperativity also destabilizes the native structure because it affects unfolding more than folding. We can thus define a continuous cooperativity scale that goes from the 'pliable' two-state character of slow folders to the gradual unfolding of one-state downhill, and eventually to intrinsically disordered proteins. The connection between gradual unfolding and intrinsic disorder is appealing because it suggests a conformational rheostat mechanism to explain the allosteric effects of folding coupled to binding.
Topics: Models, Molecular; Molecular Dynamics Simulation; Protein Conformation; Protein Folding; Protein Unfolding; Proteins; Structure-Activity Relationship
PubMed: 26845039
DOI: 10.1016/j.sbi.2015.12.001 -
Protein Engineering, Design & Selection... Feb 2021We review the background, theory and general equations for the analysis of equilibrium protein unfolding experiments, focusing on denaturant and heat-induced unfolding.... (Review)
Review
We review the background, theory and general equations for the analysis of equilibrium protein unfolding experiments, focusing on denaturant and heat-induced unfolding. The primary focus is on the thermodynamics of reversible folding/unfolding transitions and the experimental methods that are available for extracting thermodynamic parameters. We highlight the importance of modelling both how the folding equilibrium depends on a perturbing variable such as temperature or denaturant concentration, and the importance of modelling the baselines in the experimental observables.
Topics: Kinetics; Protein Denaturation; Protein Folding; Protein Stability; Temperature; Thermodynamics
PubMed: 33724431
DOI: 10.1093/protein/gzab002 -
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