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Journal of the American Chemical Society Apr 2022Although cold denaturation is a fundamental phenomenon common to all proteins, it can only be observed in a handful of cases where it occurs at temperatures above the...
Although cold denaturation is a fundamental phenomenon common to all proteins, it can only be observed in a handful of cases where it occurs at temperatures above the freezing point of water. Understanding the mechanisms that determine cold denaturation and the rules that permit its observation is an important challenge. A way to approach them is to be able to induce cold denaturation in an otherwise stable protein by means of mutations. Here, we studied CyaY, a relatively stable bacterial protein with no detectable cold denaturation and a high melting temperature of 54 °C. We have characterized for years the yeast orthologue of CyaY, Yfh1, a protein that undergoes cold and heat denaturation at 5 and 35 °C, respectively. We demonstrate that, by transferring to CyaY the lessons learnt from Yfh1, we can induce cold denaturation by introducing a restricted number of carefully designed mutations aimed at destabilizing the overall fold and inducing electrostatic frustration. We used molecular dynamics simulations to rationalize our findings and demonstrate the individual effects observed experimentally with the various mutants. Our results constitute the first example of rationally designed cold denaturation and demonstrate the importance of electrostatic frustration on the mechanism of cold denaturation.
Topics: Cold Temperature; Hot Temperature; Molecular Dynamics Simulation; Protein Denaturation; Proteins; Thermodynamics
PubMed: 35427450
DOI: 10.1021/jacs.1c13355 -
Cold Spring Harbor Perspectives in... Apr 2020The functional health of the proteome is determined by properties of the proteostasis network (PN) that regulates protein synthesis, folding, macromolecular assembly,... (Review)
Review
The functional health of the proteome is determined by properties of the proteostasis network (PN) that regulates protein synthesis, folding, macromolecular assembly, translocation, and degradation. In eukaryotes, the PN also integrates protein biogenesis across compartments within the cell and between tissues of metazoans for organismal health and longevity. Additionally, in metazoans, proteome stability and the functional health of proteins is optimized for development and yet declines throughout aging, accelerating the risk for misfolding, aggregation, and cellular dysfunction. Here, I describe the cell-nonautonomous regulation of organismal PN by tissue communication and cell stress-response pathways. These systems are robust from development through reproductive maturity and are genetically programmed to decline abruptly in early adulthood by repression of the heat shock response and other cell-protective stress responses, thus compromising the ability of cells and tissues to properly buffer against the cumulative stress of protein damage during aging. While the failure of multiple protein quality control processes during aging challenges cellular function and tissue health, genetic studies, and the identification of small-molecule proteostasis regulators suggests strategies that can be employed to reset the PN with potential benefit on cellular health and organismal longevity.
Topics: Aging; Animals; Caenorhabditis elegans; Fibroblasts; Gene Expression Regulation; Heat Shock Transcription Factors; Heat-Shock Proteins; Heat-Shock Response; Humans; Longevity; Protein Denaturation; Protein Folding; Proteins; Proteome; Proteostasis; Quality Control; Risk; Signal Transduction; Stress, Physiological
PubMed: 30962274
DOI: 10.1101/cshperspect.a034074 -
Biomolecules Jan 2021The bacterial flagellum and the related virulence-associated injectisome system of pathogenic bacteria utilize a type III secretion system (T3SS) to export substrate... (Review)
Review
The bacterial flagellum and the related virulence-associated injectisome system of pathogenic bacteria utilize a type III secretion system (T3SS) to export substrate proteins across the inner membrane in a proton motive force-dependent manner. The T3SS is composed of an export gate (FliPQR/FlhA/FlhB) located in the flagellar basal body and an associated soluble ATPase complex in the cytoplasm (FliHIJ). Here, we summarise recent insights into the structure, assembly and protein secretion mechanisms of the T3SS with a focus on energy transduction and protein transport across the cytoplasmic membrane.
Topics: Adenosine Triphosphatases; Bacterial Proteins; Cell Membrane; Cytoplasm; Flagella; Gram-Negative Bacteria; Hydrolysis; Membrane Proteins; Protein Denaturation; Protein Transport; Substrate Specificity; Type III Secretion Systems
PubMed: 33572887
DOI: 10.3390/biom11020186 -
Scientific Reports Nov 2023Thermal shift assay (TSA) with altered temperature has been the most widely used method for monitoring protein stability for drug research. However, there is a pressing...
Thermal shift assay (TSA) with altered temperature has been the most widely used method for monitoring protein stability for drug research. However, there is a pressing need for isothermal techniques as alternatives. This urgent demand arises from the limitations of TSA, which can sometimes provide misleading ranking of protein stability and fail to accurately reflect protein stability under physiological conditions. Although differential scanning fluorimetry has significantly improved throughput in comparison to differential scanning calorimetry and differential static light scattering throughput, all these methods exhibit moderate sensitivity. In contrast, current isothermal chemical denaturation (ICD) techniques may not offer the same throughput capabilities as TSA, but it provides more precise information about protein stability and interactions. Unfortunately, ICD also suffers from limited sensitivity, typically in micromolar range. We have developed a novel method to overcome these challenges, namely throughput and sensitivity. The novel Förster Resonance Energy Transfer (FRET)-Probe as an external probe is highly applicable to isothermal protein stability monitoring but also to conventional TSA. We have investigated ICD for multiple proteins with focus on KRAS with covalent inhibitors and three chemical denaturants performed at nanomolar protein concentration. Data showed corresponding inhibitor-induced stabilization of KRAS to those reported by nucleotide exchange assay.
Topics: Proto-Oncogene Proteins p21(ras); Protein Stability; Fluorometry; Calorimetry, Differential Scanning; Proteins; Protein Denaturation
PubMed: 37973851
DOI: 10.1038/s41598-023-46720-w -
Biochimica Et Biophysica Acta May 2016Differential scanning calorimetry is a powerful method that provides a complete thermodynamic characterization of the stability of a protein as a function of...
BACKGROUND
Differential scanning calorimetry is a powerful method that provides a complete thermodynamic characterization of the stability of a protein as a function of temperature. There are, however, circumstances that preclude a complete analysis of DSC data. The most common ones are irreversible denaturation transitions or transitions that take place at temperatures that are beyond the temperature limit of the instrument. Even for a protein that undergoes reversible thermal denaturation, the extrapolation of the thermodynamic data to lower temperatures, usually 25°C, may become unreliable due to difficulties in the determination of ΔCp.
METHODS
The combination of differential scanning calorimetry and isothermal chemical denaturation allows reliable thermodynamic analysis of protein stability under less than ideal conditions.
RESULTS AND CONCLUSIONS
This paper demonstrates how DSC can be used in combination with chemical denaturation to address three different scenarios: 1) estimation of an accurate ΔCp value for a reversible denaturation using as a test system the envelope HIV-1 glycoprotein gp120; 2) determination of the Gibbs energy of stability in the region in which thermal denaturation is irreversible using HEW lysozyme at different pH values; and, 3) determination of Gibbs energy of stability for a thermostable protein, thermolysin.
Topics: Animals; Bacillus; Bacterial Proteins; Calorimetry, Differential Scanning; Chickens; HIV Envelope Protein gp120; HIV-1; Hydrogen-Ion Concentration; Kinetics; Muramidase; Protein Denaturation; Protein Folding; Temperature; Thermodynamics; Thermolysin
PubMed: 26679422
DOI: 10.1016/j.bbagen.2015.12.003 -
Analytical Chemistry Oct 2021The loss of native structure is common in proteins. Among others, aggregation is one structural modification of particular importance as it is a major concern for the...
The loss of native structure is common in proteins. Among others, aggregation is one structural modification of particular importance as it is a major concern for the efficiency and safety of biotherapeutic proteins. Yet, recognizing the structural features associated with intermolecular bridging in a high-throughput manner remains a challenge. We combined here the use of protein microarrays spotted at a density of ca 2500 samples per cm and Fourier transform infrared (FTIR) imaging to analyze structural modifications in a set of 85 proteins characterized by widely different secondary structure contents, submitted or not to mild denaturing conditions. Multivariate curve resolution alternating least squares (MCR-ALS) was used to model a new spectral component appearing in the protein set subject to denaturing conditions. In the native protein set, 6 components were found to be sufficient to obtain good modeling of the spectra. Furthermore, their shape allowed them to be assigned to α-helix, β-sheet, and other structures. Their content in each protein was correlated with the known secondary structure, confirming these assignments. In the denatured proteins, a new component was necessary and modeled by MCR-ALS. This new component could be assigned to the intermolecular β-sheet, bridging protein molecules. MCR-ALS, therefore, unveiled a potential spectroscopic marker of protein aggregation and allowed a semiquantitative evaluation of its content. Insight into other structural rearrangements was also obtained.
Topics: Fourier Analysis; Least-Squares Analysis; Protein Array Analysis; Protein Denaturation; Protein Structure, Secondary; Spectroscopy, Fourier Transform Infrared
PubMed: 34592098
DOI: 10.1021/acs.analchem.1c01416 -
Journal of Controlled Release :... Oct 2016As the most abundant protein in mammals and a major structural component in extracellular matrix, collagen holds a pivotal role in tissue development and maintaining the... (Review)
Review
As the most abundant protein in mammals and a major structural component in extracellular matrix, collagen holds a pivotal role in tissue development and maintaining the homeostasis of our body. Persistent disruption to the balance between collagen production and degradation can cause a variety of diseases, some of which can be fatal. Collagen remodeling can lead to either an overproduction of collagen which can cause excessive collagen accumulation in organs, common to fibrosis, or uncontrolled degradation of collagen seen in degenerative diseases such as arthritis. Therefore, the ability to monitor the state of collagen is crucial for determining the presence and progression of numerous diseases. This review discusses the implications of collagen remodeling and its detection methods with specific focus on targeting native collagens as well as denatured collagens. It aims to help researchers understand the pathobiology of collagen-related diseases and create novel collagen targeting therapeutics and imaging modalities for biomedical applications.
Topics: Collagen; Diagnostic Imaging; Drug Delivery Systems; Extracellular Matrix; Humans; Peptides; Protein Binding; Protein Denaturation
PubMed: 26773768
DOI: 10.1016/j.jconrel.2016.01.007 -
ACS Applied Materials & Interfaces Oct 2023Proteins unfold in chaotropic salt solutions, a process that is difficult to observe at the single protein level. The work presented here demonstrates that a...
Proteins unfold in chaotropic salt solutions, a process that is difficult to observe at the single protein level. The work presented here demonstrates that a liquid-based atomic force microscope and graphene liquid-cell-based scanning transmission electron microscope make it possible to observe chemically induced protein unfolding. To illustrate this capability, ferritin proteins were deposited on a graphene surface, and the concentration-dependent urea- or guanidinium-induced changes of morphology were monitored for holo-ferritin with its ferrihydrite core as well as apo-ferritin without this core. Depending on the chaotropic agent the liquid-based imaging setup captured an unexpected transformation of natively folded holo-ferritin proteins into rings after urea treatment but not after guanidinium treatment. Urea treatment of apo-ferritin did not result in nanorings, confirming that nanorings are a specific signature of denaturation of holo-ferritins after exposture to sufficiently high urea concentrations. Mapping the images with molecular dynamics simulations of ferritin subunits in urea solutions suggests that electrostatic destabilization triggers denaturation of ferritin as urea makes direct contact with the protein and also disrupts the water H-bonding network in the ferritin solvation shell. Our findings deepen the understanding of protein denaturation studied using label-free techniques operating at the solid-liquid interface.
Topics: Guanidine; Protein Denaturation; Graphite; Ferritins; Urea
PubMed: 37797325
DOI: 10.1021/acsami.3c10510 -
Nature Jan 2019Specificity of interactions between two DNA strands, or between protein and DNA, is often achieved by varying bases or side chains coming off the DNA or protein...
Specificity of interactions between two DNA strands, or between protein and DNA, is often achieved by varying bases or side chains coming off the DNA or protein backbone-for example, the bases participating in Watson-Crick pairing in the double helix, or the side chains contacting DNA in TALEN-DNA complexes. By contrast, specificity of protein-protein interactions usually involves backbone shape complementarity, which is less modular and hence harder to generalize. Coiled-coil heterodimers are an exception, but the restricted geometry of interactions across the heterodimer interface (primarily at the heptad a and d positions) limits the number of orthogonal pairs that can be created simply by varying side-chain interactions. Here we show that protein-protein interaction specificity can be achieved using extensive and modular side-chain hydrogen-bond networks. We used the Crick generating equations to produce millions of four-helix backbones with varying degrees of supercoiling around a central axis, identified those accommodating extensive hydrogen-bond networks, and used Rosetta to connect pairs of helices with short loops and to optimize the remainder of the sequence. Of 97 such designs expressed in Escherichia coli, 65 formed constitutive heterodimers, and the crystal structures of four designs were in close agreement with the computational models and confirmed the designed hydrogen-bond networks. In cells, six heterodimers were fully orthogonal, and in vitro-following mixing of 32 chains from 16 heterodimer designs, denaturation in 5 M guanidine hydrochloride and reannealing-almost all of the interactions observed by native mass spectrometry were between the designed cognate pairs. The ability to design orthogonal protein heterodimers should enable sophisticated protein-based control logic for synthetic biology, and illustrates that nature has not fully explored the possibilities for programmable biomolecular interaction modalities.
Topics: Computer Simulation; DNA; Escherichia coli; Guanidine; Hydrogen Bonding; Models, Molecular; Protein Binding; Protein Denaturation; Protein Engineering; Protein Interaction Domains and Motifs; Protein Multimerization; Protein Structure, Secondary; Proteins
PubMed: 30568301
DOI: 10.1038/s41586-018-0802-y -
Biophysical Journal Aug 2021The folding stability of a protein is governed by the free-energy difference between its folded and unfolded states, which results from a delicate balance of much larger...
The folding stability of a protein is governed by the free-energy difference between its folded and unfolded states, which results from a delicate balance of much larger but almost compensating enthalpic and entropic contributions. The balance can therefore easily be shifted by an external disturbance, such as a mutation of a single amino acid or a change of temperature, in which case the protein unfolds. Effects such as cold denaturation, in which a protein unfolds because of cooling, provide evidence that proteins are strongly stabilized by the solvent entropy contribution to the free-energy balance. However, the molecular mechanisms behind this solvent-driven stability, their quantitative contribution in relation to other free-energy contributions, and how the involved solvent thermodynamics is affected by individual amino acids are largely unclear. Therefore, we addressed these questions using atomistic molecular dynamics simulations of the small protein Crambin in its native fold and a molten-globule-like conformation, which here served as a model for the unfolded state. The free-energy difference between these conformations was decomposed into enthalpic and entropic contributions from the protein and spatially resolved solvent contributions using the nonparametric method Per|Mut. From the spatial resolution, we quantified the local effects on the solvent free-energy difference at each amino acid and identified dependencies of the local enthalpy and entropy on the protein curvature. We identified a strong stabilization of the native fold by almost 500 kJ mol due to the solvent entropy, revealing it as an essential contribution to the total free-energy difference of (53 ± 84) kJ mol. Remarkably, more than half of the solvent entropy contribution arose from induced water correlations.
Topics: Entropy; Plant Proteins; Protein Conformation; Protein Denaturation; Protein Folding; Thermodynamics
PubMed: 34087209
DOI: 10.1016/j.bpj.2021.05.019