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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 -
Role of solvation effects in protein denaturation: from thermodynamics to single molecules and back.Annual Review of Physical Chemistry 2011Protein stability often is studied in vitro through the use of urea and guanidinium chloride, chemical cosolvents that disrupt protein native structure. Much controversy... (Review)
Review
Protein stability often is studied in vitro through the use of urea and guanidinium chloride, chemical cosolvents that disrupt protein native structure. Much controversy still surrounds the underlying mechanism by which these molecules denature proteins. Here we review current thinking on various aspects of chemical denaturation. We begin by discussing classic models of protein folding and how the effects of denaturants may fit into this picture through their modulation of the collapse, or coil-globule transition, which typically precedes folding. Subsequently, we examine recent molecular dynamics simulations that have shed new light on the possible microscopic origins of the solvation effects brought on by denaturants. It seems likely that both denaturants operate by facilitating solvation of hydrophobic regions of proteins. Finally, we present recent single-molecule fluorescence studies of denatured proteins, the analysis of which corroborates the role of denaturants in shifting the equilibrium of the coil-globule transition.
Topics: Fluorescence Resonance Energy Transfer; Guanidine; Hydrophobic and Hydrophilic Interactions; Molecular Dynamics Simulation; Protein Conformation; Protein Denaturation; Protein Folding; Protein Structure, Secondary; Proteins; Solvents; Thermodynamics; Urea; Water
PubMed: 21219136
DOI: 10.1146/annurev-physchem-032210-103531 -
Poultry Science Jun 2004Pale, soft, and exudative (PSE) meat is a growing problem in the turkey industry and has been associated with processing conditions such as slow carcass chilling. The...
Pale, soft, and exudative (PSE) meat is a growing problem in the turkey industry and has been associated with processing conditions such as slow carcass chilling. The development of PSE meat is caused by protein denaturation resulting from a rapid rate of pH decline early postmortem (PM) while carcass temperatures are still elevated. This research was conducted to determine the relationship of slow chilling to protein denaturation and PSE development. A total of 48 toms were conventionally processed in 2 trials at 22.5 wk of age, and chilled at 0, 10, 20, or 30 degrees C for either 45 or 90 min before deboning (at 60 or 105 min PM). Temperature and pH of the breast muscle was recorded at 15 min PM, at the time of deboning (60 or 90 min PM), and at 24 h PM. Color was determined at deboning and again at 24 h PM. Gel strength, cook loss, expressible moisture, total protein solubility, and bound phosphorylase quantities were determined on the fillets at 24 h PM. There was no difference in carcass temperature at 15 min PM, but by 105 min PM each temperature treatment was significantly different, with the carcasses chilled at 0 and 10 degrees C having the lowest temperature, the 30 degrees C-chilled birds having the highest temperature, and the 20 degrees C-chilled carcasses being intermediate but significantly different from either extreme. The carcass temperature differences at 105 min PM indicated that the carcass experienced differing chilling rates. To varying degrees, slower rates of chilling resulted in lower pH, greater degree of lightness (L* value), greater cook loss, and reduced gel strength. However, chilling rate had no effect on total protein solubility or myofibrillar phosphorylase for any of the treatments. Chilling rate seems to contribute to PSE turkey meat characteristics but by a mechanism independent of total protein solubility or myofibrillar phosphorylase.
Topics: Animals; Cold Temperature; Food Handling; Food Technology; Hydrogen-Ion Concentration; Meat; Muscle Proteins; Protein Denaturation; Turkeys
PubMed: 15206634
DOI: 10.1093/ps/83.6.1039 -
International Journal of Hyperthermia :... 1987Respiration of Chinese hamster lung V79 cells, as assayed by O2 consumption, increases linearly from 8 to 40 degrees C when plotted in the Arrhenius fashion but is...
Respiration of Chinese hamster lung V79 cells, as assayed by O2 consumption, increases linearly from 8 to 40 degrees C when plotted in the Arrhenius fashion but is strongly inhibited above 40 degrees C. The protein of mitochondria isolated from V79 cells undergoes structural transitions at 28 and 40 degrees C. This is supported by changes in the fluorescence excitation spectrum of conjugated pyrene maleimide and, to a lesser extent, intrinsic protein fluorophores. Electron spin resonance labelling studies with a derivative of tempo maleimide imply that extensive protein unfolding coincides with the 40 degrees C transition. The structural transition at 40 degrees C correlates well with inhibition of O2 consumption, is irreversible and is probably due to protein denaturation, while the change at 28 degrees C is reversible and has no effect on O2 consumption. Previous studies indicate the presence of a broad lipid transition extending from approximately 8 to 30 degrees C in mitochondrial membranes with all lipids being in the liquid-crystalline state above 30 degrees C. Thus, the onset of the lipid transition may induce the observed protein conformational change at 28 degrees C, but inhibition of respiration above 40 degrees C can be explained by protein denaturation alone. The region from 28 to 40 degrees C of stable protein conformation corresponds to the temperature range of V79 cell growth.
Topics: Animals; Calorimetry; Cell Line; Electron Spin Resonance Spectroscopy; Hyperthermia, Induced; Mitochondria; Oxygen Consumption; Protein Denaturation; Spectrometry, Fluorescence; Spin Labels
PubMed: 3036971
DOI: 10.3109/02656738709140380 -
Angewandte Chemie (International Ed. in... Aug 2017A hallmark of tissue ageing is the irreversible oxidative modification of its proteins. We show that single proteins, kept unfolded and extended by a mechanical force,...
A hallmark of tissue ageing is the irreversible oxidative modification of its proteins. We show that single proteins, kept unfolded and extended by a mechanical force, undergo accelerated ageing in times scales of minutes to days. A protein forced to be continuously unfolded completely loses its ability to contract by folding, becoming a labile polymer. Ageing rates vary among different proteins, but in all cases they lose their mechanical integrity. Random oxidative modification of cryptic side chains exposed by mechanical unfolding can be slowed by the addition of antioxidants such as ascorbic acid, or accelerated by oxidants. By contrast, proteins kept in the folded state and probed over week-long experiments show greatly reduced rates of ageing. We demonstrate a novel approach whereby protein ageing can be greatly accelerated: the constant unfolding of a protein for hours to days is equivalent to decades of exposure to free radicals under physiological conditions.
Topics: Antioxidants; Mechanical Phenomena; Protein Denaturation; Protein Folding; Proteins
PubMed: 28470663
DOI: 10.1002/anie.201703630 -
Protein Science : a Publication of the... Oct 2009The effects of temperature and urea denaturation (6M urea) on the dominant structures of the 20-residue Trp-cage mini-protein TC5b are investigated by molecular dynamics...
The effects of temperature and urea denaturation (6M urea) on the dominant structures of the 20-residue Trp-cage mini-protein TC5b are investigated by molecular dynamics simulations of the protein at different temperatures in aqueous and in 6M urea solution using explicit solvent degrees of freedom and the GROMOS force-field parameter set 45A3. In aqueous solution at 278 K, TC5b is stable throughout the 20 ns of MD simulation and the trajectory structures largely agree with the NMR-NOE atom-atom distance data available. Raising the temperature to 360 K and to 400 K, the protein denatures within 22 ns and 3 ns, showing that the denaturation temperature is well below 360 K using the GROMOS force field. This is 40-90 K lower than the denaturation temperatures observed in simulations using other much used protein force fields. As the experimental denaturation temperature is about 315 K, the GROMOS force field appears not to overstabilize TC5b, as other force fields and the use of continuum solvation models seem to do. This feature may directly stem from the GROMOS force-field parameter calibration protocol, which primarily involves reproduction of condensed phase thermodynamic quantities such as energies, densities, and solvation free energies of small compounds representative for protein fragments. By adding 6M urea to the solution, the onset of denaturation is observed in the simulation, but is too slow to observe a particular side-chain side-chain contact (Trp6-Ile4) that was experimentally observed to be characteristic for the denatured state. Interestingly, using temperature denaturation, the process is accelerated and the experimental data are reproduced.
Topics: Peptides; Protein Conformation; Protein Denaturation; Recombinant Proteins; Temperature; Thermodynamics; Urea
PubMed: 19693803
DOI: 10.1002/pro.223 -
Molecules (Basel, Switzerland) Jul 2022Quite a long time ago, Oleg B. Ptitsyn put forward a hypothesis about the possible functional significance of the molten globule (MG) state for the functioning of... (Review)
Review
Quite a long time ago, Oleg B. Ptitsyn put forward a hypothesis about the possible functional significance of the molten globule (MG) state for the functioning of proteins. MG is an intermediate between the unfolded and the native state of a protein. Its experimental detection and investigation in a cell are extremely difficult. In the last decades, intensive studies have demonstrated that the MG-like state of some globular proteins arises from either their modifications or interactions with protein partners or other cell components. This review summarizes such reports. In many cases, MG was evidenced to be functionally important. Thus, the MG state is quite common for functional cellular proteins. This supports Ptitsyn's hypothesis that some globular proteins may switch between two active states, rigid (N) and soft (MG), to work in solution or interact with partners.
Topics: Circular Dichroism; Protein Conformation; Protein Denaturation; Protein Folding; Proteins
PubMed: 35889244
DOI: 10.3390/molecules27144361 -
International Journal of Molecular... Jun 2021Human phenylalanine hydroxylase (PAH) is a metabolic enzyme involved in the catabolism of L-Phe in liver. Loss of conformational stability and decreased enzymatic...
Human phenylalanine hydroxylase (PAH) is a metabolic enzyme involved in the catabolism of L-Phe in liver. Loss of conformational stability and decreased enzymatic activity in PAH variants result in the autosomal recessive disorder phenylketonuria (PKU), characterized by developmental and psychological problems if not treated early. One current therapeutic approach to treat PKU is based on pharmacological chaperones (PCs), small molecules that can displace the folding equilibrium of unstable PAH variants toward the native state, thereby rescuing the physiological function of the enzyme. Understanding the PAH folding equilibrium is essential to develop new PCs for different forms of the disease. We investigate here the urea and the thermal-induced denaturation of full-length PAH and of a truncated form lacking the regulatory and the tetramerization domains. For either protein construction, two distinct transitions are seen in chemical denaturation followed by fluorescence emission, indicating the accumulation of equilibrium unfolding intermediates where the catalytic domains are partly unfolded and dissociated from each other. According to analytical centrifugation, the chemical denaturation intermediates of either construction are not well-defined species but highly polydisperse ensembles of protein aggregates. On the other hand, each protein construction similarly shows two transitions in thermal denaturation measured by fluorescence or differential scanning calorimetry, also indicating the accumulation of equilibrium unfolding intermediates. The similar temperatures of mid denaturation of the two constructions, together with their apparent lack of response to protein concentration, indicate the catalytic domains are unfolded in the full-length PAH thermal intermediate, where they remain associated. That the catalytic domain unfolds in the first thermal transition is relevant for the choice of PCs identified in high throughput screening of chemical libraries using differential scanning fluorimetry.
Topics: Binding Sites; Calorimetry, Differential Scanning; Catalytic Domain; Humans; Molecular Docking Simulation; Molecular Dynamics Simulation; Phenylalanine Hydroxylase; Phenylketonurias; Protein Conformation; Protein Denaturation; Protein Folding; Protein Stability; Temperature; Thermodynamics; Urea
PubMed: 34207146
DOI: 10.3390/ijms22126539 -
Traffic (Copenhagen, Denmark) Jun 2002Diverse human diseases ranging from amyloidosis to neurodegenerative diseases are now recognized as 'conformational diseases' caused by protein misfolding and protein... (Review)
Review
Diverse human diseases ranging from amyloidosis to neurodegenerative diseases are now recognized as 'conformational diseases' caused by protein misfolding and protein aggregation. Misfolded and aggregated proteins are usually handled in the cell through chaperone-mediated refolding, or when that is impossible, destroyed by proteasomal degradation. Recent evidence suggests that cells might have evolved a third pathway that involves the sequestration of aggregated proteins into specialized 'holding stations' called aggresomes. The aggresomal pathway provides a mechanism by which aggregated proteins form particulate (approximately 200 nm) mini-aggregates that are transported on microtubules (MTs) towards the MT organizing center (MTOC) by a process mediated by the minus-end motor protein dynein. Once at the MTOC, the individual particles pack into a single, usually spherical aggresome (1-3 microm) that surrounds the MTOC. Aggresomes are dynamic: they recruit various chaperones and proteasomes, presumably to aid in the disposal of the aggregated proteins. In addition, the formation of an aggresome is likely to activate the autophagic clearance mechanism that terminates in lysosomal degradation. Hence, the aggresome pathway may provide a novel system to deliver aggregated proteins from the cytoplasm to lysosomes for degradation. Although it is clear that many pathological states correlate with the formation of aggresomes, their causal relationships remain hotly debated. Here, we describe the current state of our knowledge of the aggresome pathway and outline the open questions that provide the focus of current research.
Topics: Cytoskeleton; Disease; Humans; Lysosomes; Protein Denaturation; Protein Folding
PubMed: 12010457
DOI: 10.1034/j.1600-0854.2002.30602.x -
International Journal of Molecular... Sep 2018Near-infrared fluorescent proteins (NIR FPs) based on the complexes of bacterial phytochromes with their natural biliverdin chromophore are widely used as genetically...
Near-infrared fluorescent proteins (NIR FPs) based on the complexes of bacterial phytochromes with their natural biliverdin chromophore are widely used as genetically encoded optical probes for visualization of cellular processes and deep-tissue imaging of cells and organs in living animals. In this work, we show that the steady-state and kinetic dependencies of the various spectral characteristics of iRFP713, developed from the bacterial phytochrome BphP2 and recorded at protein unfolding induced by guanidine hydrochloride (GdnHCl), guanidine thiocyanate (GTC), and urea, differ substantially. A study of the unfolding of three single-tryptophan mutant forms of iRFP713 expectedly revealed that protein unfolding begins with the dissociation of the native dimer, while the monomers remain compact. A further increase in the denaturant concentration leads to the formation of an intermediate state of iRFP713 having hydrophobic areas exposed on the protein surface (I). The total surface charge of iRFP713 (pI 5.86) changes from negative to positive with an increase in the concentration of GdnHCl and GTC because the negative charge of glutamic and aspartic acids is neutralized by forming salt bridges between the carboxyl groups and GdnH⁺ ions and because the guanidinium cations bind to amide groups of glutamines and asparagines. The coincidence of both the concentration of the denaturants at which the intermediate state of iRFP713 accumulates and the concentration of GdnH⁺ ions at which the neutralization of the surface charge of the protein in this state is ensured results in strong protein aggregation. This is evidently realized by iRFP713 unfolding by GTC. At the unfolding of the protein by GdnHCl, an intermediate state is populated at higher denaturant concentrations and a strong aggregation is not observed. As expected, protein aggregates are not formed in the presence of the urea. The aggregation of the protein upon neutralization of the charge on the macromolecule surface is the main indicator of the intermediate state of protein. The unfolded state of iRFP713, whose formation is accompanied by a significant decrease in the parameter , was found to have a different residual structure in the denaturants used.
Topics: Guanidine; Guanidines; Kinetics; Luminescent Proteins; Protein Aggregates; Protein Denaturation; Protein Folding; Protein Unfolding; Thiocyanates
PubMed: 30223568
DOI: 10.3390/ijms19092776