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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 -
Progress in Nuclear Magnetic Resonance... May 2017Protein folding is a highly complex process proceeding through a number of disordered and partially folded nonnative states with various degrees of structural... (Review)
Review
Protein folding is a highly complex process proceeding through a number of disordered and partially folded nonnative states with various degrees of structural organization. These transiently and sparsely populated species on the protein folding energy landscape play crucial roles in driving folding toward the native conformation, yet some of these nonnative states may also serve as precursors for protein misfolding and aggregation associated with a range of devastating diseases, including neuro-degeneration, diabetes and cancer. Therefore, in vivo protein folding is often reshaped co- and post-translationally through interactions with the ribosome, molecular chaperones and/or other cellular components. Owing to developments in instrumentation and methodology, solution NMR spectroscopy has emerged as the central experimental approach for the detailed characterization of the complex protein folding processes in vitro and in vivo. NMR relaxation dispersion and saturation transfer methods provide the means for a detailed characterization of protein folding kinetics and thermodynamics under native-like conditions, as well as modeling high-resolution structures of weakly populated short-lived conformational states on the protein folding energy landscape. Continuing development of isotope labeling strategies and NMR methods to probe high molecular weight protein assemblies, along with advances of in-cell NMR, have recently allowed protein folding to be studied in the context of ribosome-nascent chain complexes and molecular chaperones, and even inside living cells. Here we review solution NMR approaches to investigate the protein folding energy landscape, and discuss selected applications of NMR methodology to studying protein folding in vitro and in vivo. Together, these examples highlight a vast potential of solution NMR in providing atomistic insights into molecular mechanisms of protein folding and homeostasis in health and disease.
Topics: Humans; Magnetic Resonance Spectroscopy; Models, Molecular; Molecular Chaperones; Protein Binding; Protein Conformation; Protein Denaturation; Protein Domains; Protein Folding; Proteins; Ribosomes; Thermodynamics
PubMed: 28552172
DOI: 10.1016/j.pnmrs.2016.10.002 -
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 -
International Journal of Molecular... Feb 2024Proteins are large biomolecules with a specific structure that is composed of one or more long amino acid chains. Correct protein structures are directly linked to their... (Review)
Review
Proteins are large biomolecules with a specific structure that is composed of one or more long amino acid chains. Correct protein structures are directly linked to their correct function, and many environmental factors can have either positive or negative effects on this structure. Thus, there is a clear need for methods enabling the study of proteins, their correct folding, and components affecting protein stability. There is a significant number of label-free methods to study protein stability. In this review, we provide a general overview of these methods, but the main focus is on fluorescence-based low-instrument and -expertise-demand techniques. Different aspects related to thermal shift assays (TSAs), also called differential scanning fluorimetry (DSF) or ThermoFluor, are introduced and compared to isothermal chemical denaturation (ICD). Finally, we discuss the challenges and comparative aspects related to these methods, as well as future opportunities and assay development directions.
Topics: Protein Stability; Proteins; Amino Acids; Fluorometry; Biological Assay; Protein Denaturation
PubMed: 38339045
DOI: 10.3390/ijms25031764 -
Journal of the Royal Society, Interface Oct 2018Although it is now relatively well understood how sequence defines and impacts global protein stability in specific structural contexts, the question of how sequence... (Review)
Review
Although it is now relatively well understood how sequence defines and impacts global protein stability in specific structural contexts, the question of how sequence modulates the configurational landscape of proteins remains to be defined. Protein configurational equilibria are generally characterized by using various chemical denaturants or by changing temperature or pH. Another thermodynamic parameter which is less often used in such studies is high hydrostatic pressure. This review discusses the basis for pressure effects on protein structure and stability, and describes how the unique mechanisms of pressure-induced unfolding can provide unique insights into protein conformational landscapes.
Topics: Animals; Pressure; Protein Denaturation; Protein Stability; Proteins; Thermodynamics
PubMed: 30282759
DOI: 10.1098/rsif.2018.0244 -
International Journal of Molecular... Jun 2021Oxidative stress, photo-oxidation, and photosensitizers are activated by UV irradiation and are affecting the photo-stability of proteins. Understanding the mechanisms...
Oxidative stress, photo-oxidation, and photosensitizers are activated by UV irradiation and are affecting the photo-stability of proteins. Understanding the mechanisms that govern protein photo-stability is essential for its control enabling enhancement or reduction. Currently, two major mechanisms for protein denaturation induced by UV irradiation are available: one generated by the local heating of water molecules bound to the proteins and the other by the formation of reactive free radicals. To discriminate which is the likely or dominant mechanism we have studied the effects of thermal and UV denaturation of aqueous protein solutions with and without DHR-123 as fluorogenic probe using circular dichroism (CD), synchrotron radiation circular dichroism (SRCD), and fluorescence spectroscopies. The results indicated that the mechanism of protein denaturation induced by VUV and far-UV irradiation were mediated by the formation of reactive free radicals (FR) and reactive oxygen species (ROS). The development at Diamond B23 beamline for SRCD of a novel protein UV photo-stability assay based on consecutive repeated CD measurements in the far-UV (180-250 nm) region has been successfully used to assess and characterize the photo-stability of protein formulations and ligand binding interactions, in particular for ligand molecules devoid of significant UV absorption.
Topics: Circular Dichroism; Free Radicals; Heating; Protein Denaturation; Proteins; Reactive Oxygen Species; Spectrum Analysis; Ultraviolet Rays; Water
PubMed: 34204483
DOI: 10.3390/ijms22126512 -
Cold Spring Harbor Perspectives in... Jan 2020Cells invest in an extensive network of factors to maintain protein homeostasis (proteostasis) and prevent the accumulation of potentially toxic protein aggregates. This... (Review)
Review
Cells invest in an extensive network of factors to maintain protein homeostasis (proteostasis) and prevent the accumulation of potentially toxic protein aggregates. This proteostasis network (PN) comprises the machineries for the biogenesis, folding, conformational maintenance, and degradation of proteins with molecular chaperones as central coordinators. Here, we review recent progress in understanding the modular architecture of the PN in mammalian cells and how it is modified during cell differentiation. We discuss the capacity and limitations of the PN in maintaining proteome integrity in the face of proteotoxic stresses, such as aggregate formation in neurodegenerative diseases. Finally, we outline various pharmacological interventions to ameliorate proteostasis imbalance.
Topics: Animals; Cell Differentiation; Homeostasis; Humans; Molecular Chaperones; Neurodegenerative Diseases; Protein Conformation; Protein Denaturation; Protein Folding; Proteins; Proteome; Proteostasis; Thermodynamics
PubMed: 30833457
DOI: 10.1101/cshperspect.a033951 -
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 -
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 -
Quarterly Reviews of Biophysics Feb 2020Proteins are molecular machines whose function depends on their ability to achieve complex folds with precisely defined structural and dynamic properties. The rational... (Review)
Review
Proteins are molecular machines whose function depends on their ability to achieve complex folds with precisely defined structural and dynamic properties. The rational design of proteins from first-principles, or de novo, was once considered to be impossible, but today proteins with a variety of folds and functions have been realized. We review the evolution of the field from its earliest days, placing particular emphasis on how this endeavor has illuminated our understanding of the principles underlying the folding and function of natural proteins, and is informing the design of macromolecules with unprecedented structures and properties. An initial set of milestones in de novo protein design focused on the construction of sequences that folded in water and membranes to adopt folded conformations. The first proteins were designed from first-principles using very simple physical models. As computers became more powerful, the use of the rotamer approximation allowed one to discover amino acid sequences that stabilize the desired fold. As the crystallographic database of protein structures expanded in subsequent years, it became possible to construct proteins by assembling short backbone fragments that frequently recur in Nature. The second set of milestones in de novo design involves the discovery of complex functions. Proteins have been designed to bind a variety of metals, porphyrins, and other cofactors. The design of proteins that catalyze hydrolysis and oxygen-dependent reactions has progressed significantly. However, de novo design of catalysts for energetically demanding reactions, or even proteins that bind with high affinity and specificity to highly functionalized complex polar molecules remains an importnant challenge that is now being achieved. Finally, the protein design contributed significantly to our understanding of membrane protein folding and transport of ions across membranes. The area of membrane protein design, or more generally of biomimetic polymers that function in mixed or non-aqueous environments, is now becoming increasingly possible.
Topics: Amino Acid Motifs; Animals; Binding Sites; Biotechnology; Catalysis; Crystallography, X-Ray; Humans; Hydrogen Bonding; Ions; Kinetics; Ligands; Macromolecular Substances; Protein Binding; Protein Denaturation; Protein Engineering; Protein Folding; Proteins; Zinc
PubMed: 32041676
DOI: 10.1017/S0033583519000131