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Structure (London, England : 1993) Jan 2017Kinetic stability is a key parameter to comprehend protein behavior and it plays a central role to understand how evolution has reached the balance between function and...
Kinetic stability is a key parameter to comprehend protein behavior and it plays a central role to understand how evolution has reached the balance between function and stability in cell-relevant timescales. Using an approach that includes simulations, protein engineering, and calorimetry, we show that there is a clear correlation between kinetic stability determined by differential scanning calorimetry and protein thermal flexibility obtained from a novel method based on temperature-induced unfolding molecular dynamics simulations. Thermal flexibility quantitatively measures the increment of the conformational space available to the protein when energy in provided. The (β/α) barrel fold of two closely related by evolution triosephosphate isomerases from two trypanosomes are used as model systems. The kinetic stability-thermal flexibility correlation has predictive power for the studied proteins, suggesting that the strategy and methodology discussed here might be applied to other proteins in biotechnological developments, evolutionary studies, and the design of protein based therapeutics.
Topics: Calorimetry, Differential Scanning; Kinetics; Molecular Conformation; Protein Denaturation; Protein Engineering; Protein Stability; Protein Structure, Secondary; Protein Unfolding; Thermodynamics; Triose-Phosphate Isomerase; Trypanosoma
PubMed: 28052236
DOI: 10.1016/j.str.2016.11.018 -
Biophysical Journal Jun 2021The relationship between the dimensions of pressure-unfolded states of proteins compared with those at ambient pressure is controversial; resolving this issue is related...
The relationship between the dimensions of pressure-unfolded states of proteins compared with those at ambient pressure is controversial; resolving this issue is related directly to the mechanisms of pressure denaturation. Moreover, a significant pressure dependence of the compactness of unfolded states would complicate the interpretation of folding parameters from pressure perturbation and make comparison to those obtained using alternative perturbation approaches difficult. Here, we determined the compactness of the pressure-unfolded state of a small, cooperatively folding model protein, CTL9-I98A, as a function of temperature. This protein undergoes both thermal unfolding and cold denaturation, and the temperature dependence of the compactness at atmospheric pressure is known. High-pressure small angle x-ray scattering studies, yielding the radius of gyration and high-pressure diffusion ordered spectroscopy NMR experiments, yielding the hydrodynamic radius were carried out as a function of temperature at 250 MPa, a pressure at which the protein is unfolded. The radius of gyration values obtained at any given temperature at 250 MPa were similar to those reported previously at ambient pressure, and the trends with temperature are similar as well, although the pressure-unfolded state appears to undergo more pronounced expansion at high temperature than the unfolded state at atmospheric pressure. At 250 MPa, the compaction of the unfolded chain was maximal between 25 and 30°C, and the chain expanded upon both cooling and heating. These results reveal that the pressure-unfolded state of this protein is very similar to that observed at ambient pressure, demonstrating that pressure perturbation represents a powerful approach for observing the unfolded states of proteins under otherwise near-native conditions.
Topics: Cold Temperature; Protein Conformation; Protein Denaturation; Protein Folding; Ribosomal Proteins; Temperature
PubMed: 33961866
DOI: 10.1016/j.bpj.2021.04.031 -
Biomolecules Mar 2020From experimental studies of protein folding, it is now clear that there are two types of folding behavior, i.e., two-state folding and non-two-state folding, and... (Review)
Review
From experimental studies of protein folding, it is now clear that there are two types of folding behavior, i.e., two-state folding and non-two-state folding, and understanding the relationships between these apparently different folding behaviors is essential for fully elucidating the molecular mechanisms of protein folding. This article describes how the presence of the two types of folding behavior has been confirmed experimentally, and discusses the relationships between the two-state and the non-two-state folding reactions, on the basis of available data on the correlations of the folding rate constant with various structure-based properties, which are determined primarily by the backbone topology of proteins. Finally, a two-stage hierarchical model is proposed as a general mechanism of protein folding. In this model, protein folding occurs in a hierarchical manner, reflecting the hierarchy of the native three-dimensional structure, as embodied in the case of non-two-state folding with an accumulation of the molten globule state as a folding intermediate. The two-state folding is thus merely a simplified version of the hierarchical folding caused either by an alteration in the rate-limiting step of folding or by destabilization of the intermediate.
Topics: Circular Dichroism; Kinetics; Protein Conformation; Protein Denaturation; Protein Folding; Proteins
PubMed: 32155758
DOI: 10.3390/biom10030407 -
Biophysical Journal Apr 2022Yfh1 is a yeast protein with the peculiar characteristic to undergo, in the absence of salt, cold denaturation at temperatures above the water freezing point. This...
Yfh1 is a yeast protein with the peculiar characteristic to undergo, in the absence of salt, cold denaturation at temperatures above the water freezing point. This feature makes the protein particularly interesting for studies aiming at understanding the rules that determine protein fold stability. Here, we present the phase diagram of Yfh1 unfolding as a function of pressure (0.1-500 MPa) and temperature 278-313 K (5-40°C) both in the absence and in the presence of stabilizers using Trp fluorescence as a monitor. The protein showed a remarkable sensitivity to pressure: at 293 K, pressures around 10 MPa are sufficient to cause 50% of unfolding. Higher pressures were required for the unfolding of the protein in the presence of stabilizers. The phase diagram on the pressure-temperature plane together with a critical comparison between our results and those found in the literature allowed us to draw conclusions on the mechanism of the unfolding process under different environmental conditions.
Topics: Cold Temperature; Hot Temperature; Iron-Binding Proteins; Protein Denaturation; Protein Folding; Saccharomyces cerevisiae; Thermodynamics; Frataxin
PubMed: 35278425
DOI: 10.1016/j.bpj.2022.03.010 -
Biochimica Et Biophysica Acta Jan 2016It is probable that the great majority of human cataract results from the spontaneous decomposition of long-lived macromolecules in the human lens. Breakdown/reaction of... (Review)
Review
BACKGROUND
It is probable that the great majority of human cataract results from the spontaneous decomposition of long-lived macromolecules in the human lens. Breakdown/reaction of long-lived proteins is of primary importance and recent proteomic analysis has enabled the identification of the particular crystallins, and their exact sites of amino acid modification.
SCOPE OF REVIEW
Analysis of proteins from cataractous lenses revealed that there are sites on some structural proteins that show a consistently greater degree of deterioration than age-matched normal lenses.
MAJOR CONCLUSIONS
The most abundant posttranslational modification of aged lens proteins is racemization. Deamidation, truncation and crosslinking, each arising from the spontaneous breakdown of susceptible amino acids within proteins, are also present. Fundamental to an understanding of nuclear cataract etiology, it is proposed that once a certain degree of modification at key sites occurs, that protein-protein interactions are disrupted and lens opacification ensues.
GENERAL SIGNIFICANCE
Since long-lived proteins are now recognized to be present in many other sites of the body, such as the brain, the information gleaned from detailed analyses of degraded proteins from aged lenses will apply more widely to other age-related human diseases. This article is part of a Special Issue entitled Crystallin Biochemistry in Health and Disease.
Topics: Aging; Animals; Cataract; Crystallins; Humans; Lens, Crystalline; Models, Biological; Models, Chemical; Protein Denaturation; Protein Folding
PubMed: 26318017
DOI: 10.1016/j.bbagen.2015.08.016 -
Protein and Peptide Letters 2022The analysis of biofluid samples with low protein content (e.g., urine or saliva) can be challenging for downstream analysis methods with limited sensitivity. To...
BACKGROUND
The analysis of biofluid samples with low protein content (e.g., urine or saliva) can be challenging for downstream analysis methods with limited sensitivity. To circumvent this problem, sample processing methods are employed to increase the protein concentration in analyzed samples. However, for some techniques, like differential scanning calorimetry (DSC) that characterizes thermally-induced unfolding of biomolecules, sample processing must not affect native protein structure and stability.
METHODS
We evaluated centrifugal concentration and stirred cell ultrafiltration, two common methods of sample concentration characterized by a low risk of protein denaturation, with the goal of establishing a protocol for DSC analysis of low concentration biospecimens.
RESULTS
Our studies indicate that both methods can affect protein stability assessed by DSC and, even after optimization of several parameters, the obtained DSC profile (thermogram) suggested that sample processing affects the structure or intermolecular interactions of component proteins contributing to altered thermal stability detectable by DSC. We also found a relationship between changes in thermograms and low protein concentration, indicating that diluting biospecimens to concentrations below 0.1 mg/mL can perturb the intermolecular environment and affect the structure of proteins present in the solution.
CONCLUSION
Dilution of samples below 0.1 mg/mL, as well as concentration of samples with low protein content, resulted in affected thermogram shapes suggesting changes in protein stability. This should be taken into account when concentrating dilute samples or employing techniques that lower the protein concentration (e.g., fractionation), when downstream applications include techniques, such as DSC, that require the preservation of native protein forms.
Topics: Calorimetry, Differential Scanning; Protein Denaturation; Protein Stability; Proteins; Specimen Handling
PubMed: 35430965
DOI: 10.2174/0929866529666220416164305 -
International Journal of Molecular... Mar 2022The folding of lysozyme in glycerol was monitored by the fast scanning calorimetry technique. Application of a temperature-time profile with an isothermal segment for...
The folding of lysozyme in glycerol was monitored by the fast scanning calorimetry technique. Application of a temperature-time profile with an isothermal segment for refolding allowed assessment of the state of the non-equilibrium protein ensemble and gave information on the kinetics of folding. We found that the non-equilibrium protein ensemble mainly contains a mixture of unfolded and folded protein forms and partially folded intermediates, and enthalpic barriers control the kinetics of the process. Lysozyme folding in glycerol follows the same or similar triangular mechanism described in the literature for folding in water. The unfolding enthalpy of the intermediate must be no lower than 70% of the folded form, while the activation barrier for the unfolding of the intermediate (ca. 140 kJ/mol) is about 100 kJ/mol lower than that of the folded form (ca. 240-260 kJ/mol).
Topics: Calorimetry; Glycerol; Hydrogen-Ion Concentration; Kinetics; Muramidase; Protein Denaturation; Protein Folding; Proteins; Thermodynamics
PubMed: 35269914
DOI: 10.3390/ijms23052773 -
Molecules (Basel, Switzerland) Jul 2018Light irradiation with high photon flux in the vacuum and far-UV region is known to denature the conformation of biopolymers. Measures are in place at Diamond Light... (Review)
Review
Light irradiation with high photon flux in the vacuum and far-UV region is known to denature the conformation of biopolymers. Measures are in place at Diamond Light Source B23 beamline for Synchrotron Radiation Circular Dichroism (SRCD) to control and make this effect negligible. However, UV denaturation of proteins can also be exploited as a novel method for assessing biopolymer photostability as well as ligand-binding interactions. Usually, host⁻ligand binding interactions can be assessed monitoring CD changes of the host biopolymer upon ligand addition. The novel method of identifying ligand binding monitoring the change of relative rate of UV denaturation using SRCD is especially important when there are very little or insignificant secondary structure changes of the host protein upon ligand binding. The temperature study, another method used to determine molecular interactions, can often be inconclusive when the thermal effect associated with the displacement of the bound solvent molecules by the ligand is also small, making the determination of the binding interaction inconclusive. Herein we present a review on the UV-denaturation assay as a novel method to determine the relative photostability of protein formulations as well as the screening of ligand-binding interactions using the high photon flux Diamond B23 beamline for SRCD.
Topics: Biological Assay; Ceftriaxone; Circular Dichroism; Glial Fibrillary Acidic Protein; Gold; Humans; Ligands; Metal Nanoparticles; Phenytoin; Photons; Protein Binding; Protein Denaturation; Protein Stability; Protein Structure, Secondary; Serum Albumin, Human; Synchrotrons; Ultraviolet Rays
PubMed: 30065161
DOI: 10.3390/molecules23081906 -
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 -
ACS Chemical Biology Sep 2021Membrane protein structures provide a fundamental understanding of their molecular actions and are of importance for drug development. Detergents are widely used to...
Membrane protein structures provide a fundamental understanding of their molecular actions and are of importance for drug development. Detergents are widely used to solubilize, stabilize, and crystallize membrane proteins, but membrane proteins solubilized in conventional detergents are prone to denaturation and aggregation. Thus, developing novel detergents with enhanced efficacy for protein stabilization remains important. We report herein the design and synthesis of a class of phenol-derived maltoside detergents. Using two different linkers, we prepared two sets of new detergents, designated maltose-bis(hydroxymethyl)phenol (MBPs) and maltose-tris(hydroxymethyl)phenol (MTPs). The evaluation of these detergents with three transporters and two G-protein coupled receptors allowed us to identify a couple of new detergents (MBP-C9 and MTP-C12) that consistently conferred enhanced stability to all tested proteins compared to a gold standard detergent (DDM). Furthermore, the data analysis based on the detergent structures provides key detergent features responsible for membrane protein stabilization that together will facilitate the future design of novel detergents.
Topics: Detergents; Glycolipids; Hydrophobic and Hydrophilic Interactions; Membrane Transport Proteins; Phenol; Protein Conformation; Protein Denaturation; Protein Stability; Receptors, G-Protein-Coupled; Structure-Activity Relationship; Thermodynamics
PubMed: 34445864
DOI: 10.1021/acschembio.1c00578