-
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 -
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 -
Protein Science : a Publication of the... Sep 2021Seventy years ago, we learned from Chris Anfinsen that the stereochemical code necessary to fold a protein is embedded into its amino acid sequence. In water, protein... (Review)
Review
Seventy years ago, we learned from Chris Anfinsen that the stereochemical code necessary to fold a protein is embedded into its amino acid sequence. In water, protein morphogenesis is a spontaneous reversible process leading from an ensemble of disordered structures to the ordered functionally competent protein; conforming to Aristotle's definition of substance, the synolon of matter and form. The overall process of folding is generally consistent with a two state transition between the native and the denatured protein: not only the denatured state is an ensemble of several structures, but also the native protein populates distinct functionally relevant conformational (sub)states. This two-state view should be revised, given that any globular protein can populate a peculiar third state called amyloid, characterized by an overall architecture that at variance with the native state, is by-and-large independent of the primary structure. In a nut shell, we should accept that beside the folded and unfolded states, any protein can populate a third state called amyloid which gained center stage being the hallmark of incurable neurodegenerative disorders, such as Alzheimer's and Parkinson's diseases as well as others. These fatal diseases are characterized by clear-cut clinical differences, yet display some commonalities such as the presence in the brain of amyloid deposits constituted by one misfolded protein specific for each disease. Some aspects of this complex problem are summarized here as an excursus from the prion's fibrils observed in the brain of aborigines who died of Kuru to the amyloid detectable in the cortex of Alzheimer's patients.
Topics: Alzheimer Disease; Amyloid; Amyloid beta-Protein Precursor; Brain; Gene Expression; Humans; Kuru; Models, Molecular; Parkinson Disease; PrPC Proteins; PrPSc Proteins; Protein Conformation, alpha-Helical; Protein Conformation, beta-Strand; Protein Denaturation; Protein Folding; Thermodynamics; alpha-Synuclein; tau Proteins
PubMed: 34118168
DOI: 10.1002/pro.4145 -
Molecules (Basel, Switzerland) Nov 2019Chemical labeling of proteins with synthetic low-molecular-weight probes is an important technique in chemical biology. To achieve this, it is necessary to use chemical... (Review)
Review
Chemical labeling of proteins with synthetic low-molecular-weight probes is an important technique in chemical biology. To achieve this, it is necessary to use chemical reactions that proceed rapidly under physiological conditions (i.e., aqueous solvent, pH, low concentration, and low temperature) so that protein denaturation does not occur. The radical reaction satisfies such demands of protein labeling, and protein labeling using the biomimetic radical reaction has recently attracted attention. The biomimetic radical reaction enables selective labeling of the C-terminus, tyrosine, and tryptophan, which is difficult to achieve with conventional electrophilic protein labeling. In addition, as the radical reaction proceeds selectively in close proximity to the catalyst, it can be applied to the analysis of protein-protein interactions. In this review, recent trends in protein labeling using biomimetic radical reactions are discussed.
Topics: Biomimetic Materials; Catalysis; Free Radicals; Molecular Probes; Proteins; Staining and Labeling; Tryptophan; Tyrosine
PubMed: 31684188
DOI: 10.3390/molecules24213980 -
RSC Advances Oct 2023The entry of micro- and nanoplastics (MNPs) into the human body is inevitable. They enter blood circulation through ingestion, inhalation, and dermal contact by crossing... (Review)
Review
The entry of micro- and nanoplastics (MNPs) into the human body is inevitable. They enter blood circulation through ingestion, inhalation, and dermal contact by crossing the gut-lung-skin barrier (the epithelium of the digestive tract, the respiratory tract, and the cutaneous layer). There are many reports on their toxicities to organs and tissues. This paper presents the first thorough assessment of MNP-driven bloodstream toxicity and the mechanism of toxicity from the viewpoint of both MNP and environmental co-pollutant complexes. Toxic impacts include plasma protein denaturation, hemolysis, reduced immunity, thrombosis, blood coagulation, and vascular endothelial damage, among others, which can lead to life-threatening diseases. Protein corona formation, oxidative stress, cytokine alterations, inflammation, and cyto- and genotoxicity are the key mechanisms involved in toxicity. MNPs change the secondary structure of plasma proteins, thereby preventing their transport functions (for nutrients, drugs, oxygen, ). MNPs inhibit erythropoiesis by influencing hematopoietic stem cell proliferation and differentiation. They cause red blood cell and platelet aggregation, as well as increased adherence to endothelial cells, which can lead to thrombosis and cardiovascular disease. White blood cells and immune cells phagocytose MNPs, provoking inflammation. However, research gaps still exist, including gaps regarding the combined toxicity of MNPs and co-pollutants, toxicological studies in human models, advanced methodologies for toxicity analysis, bioaccumulation studies, inflammation and immunological responses, dose-response relationships of MNPs, and the effect of different physiochemical characteristics of MNPs. Furthermore, most studies have analyzed toxicity using prepared MNPs; hence, studies must be undertaken using true-to-life MNPs to determine the real-world scenario. Additionally, nanoplastics may further degrade into monomers, whose toxic effects have not yet been explored. The research gaps highlighted in this review will inspire future studies on the toxicity of MNPs in the vascular/circulatory systems utilizing models to enable more reliable health risk assessment.
PubMed: 37901269
DOI: 10.1039/d3ra05620a -
The Journal of Biological Chemistry May 2020Much of our current knowledge of biological chemistry is founded in the structure-function relationship, whereby sequence determines structure that determines function.... (Review)
Review
Much of our current knowledge of biological chemistry is founded in the structure-function relationship, whereby sequence determines structure that determines function. Thus, the discovery that a large fraction of the proteome is intrinsically disordered, while being functional, has revolutionized our understanding of proteins and raised new and interesting questions. Many intrinsically disordered proteins (IDPs) have been determined to undergo a disorder-to-order transition when recognizing their physiological partners, suggesting that their mechanisms of folding are intrinsically different from those observed in globular proteins. However, IDPs also follow some of the classic paradigms established for globular proteins, pointing to important similarities in their behavior. In this review, we compare and contrast the folding mechanisms of globular proteins with the emerging features of binding-induced folding of intrinsically disordered proteins. Specifically, whereas disorder-to-order transitions of intrinsically disordered proteins appear to follow rules of globular protein folding, such as the cooperative nature of the reaction, their folding pathways are remarkably more malleable, due to the heterogeneous nature of their folding nuclei, as probed by analysis of linear free-energy relationship plots. These insights have led to a new model for the disorder-to-order transition in IDPs termed "templated folding," whereby the binding partner dictates distinct structural transitions to product, while ensuring a cooperative folding.
Topics: Intrinsically Disordered Proteins; Models, Molecular; Protein Folding
PubMed: 32253236
DOI: 10.1074/jbc.REV120.012413