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The FEBS Journal Mar 2022Folding stability is a crucial feature of protein evolution and is essential for protein functions. Thus, the comprehension of protein folding mechanisms represents an...
Folding stability is a crucial feature of protein evolution and is essential for protein functions. Thus, the comprehension of protein folding mechanisms represents an important complement to protein structure and function, crucial to determine the structural basis of protein misfolding. In this context, thermal unfolding studies represent a useful tool to get a molecular description of the conformational transitions governing the folding/unfolding equilibrium of a given protein. Here, we report the thermal folding/unfolding pathway of VEGFR1D2, a member of the immunoglobulin superfamily by means of a high-resolution thermodynamic approach that combines differential scanning calorimetry with atomic-level unfolding monitored by NMR. We show how VEGFR1D2 folding is driven by an oxidatively induced disulfide pairing: the key event in the achievement of its functional structure is the formation of a small hydrophobic core that surrounds a disulfide bridge. Such a 'folding nucleus' induces the cooperative transition to the properly folded conformation supporting the hypothesis that a disulfide bond can act as a folding nucleus that eases the folding process.
Topics: Calorimetry, Differential Scanning; Circular Dichroism; Disulfides; Humans; Protein Denaturation; Protein Folding; Proteins; Thermodynamics
PubMed: 34689403
DOI: 10.1111/febs.16246 -
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 -
Molecules (Basel, Switzerland) Dec 2022Ornamental plants often gain relevance not only for their decorative use, but also as a source of phytochemicals with interesting healing properties. Herein, spontaneous...
(L.) DC. and L.: Phytochemical Profile, In Vitro Anti-Denaturation Effects and Lipase Inhibitory Activity of Two Ornamental Plants Traditionally Used as Herbal Remedies.
Ornamental plants often gain relevance not only for their decorative use, but also as a source of phytochemicals with interesting healing properties. Herein, spontaneous (L.) DC. and L., mainly used as ornamental species but also traditionally consumed and used in popular medicine, were investigated. The aerial parts were extracted with methanol trough maceration, and resultant crude extracts were partitioned using solvents with increasing polarity. As previous studies mostly dealt with the phenolic content of these species, the phytochemical investigation mainly focused on nonpolar constituents, detected with GC-MS. The total phenolic and flavonoid content was also verified, and HPTLC analyses were performed. In order to explore the potential antiarthritic and anti-obesity properties, extracts and their fractions were evaluated for their anti-denaturation effects, with the use of the BSA assay, and for their ability to inhibit pancreatic lipase. The antioxidant properties and the inhibitory activity on the NO production were verified, as well. Almost all the extracts and fractions demonstrated good inhibitory effects on NO production. The -hexane and dichloromethane fractions from , as well as the -hexane fraction from , were effective in protecting the protein from heat-induced denaturation (IC = 154.0 ± 1.9, 270.8 ± 2.3 and 450.1 ± 15.5 μg/mL, respectively). The dichloromethane fractions from both raw extracts were also effective in inhibiting pancreatic lipase, with IC values equal to 2.23 ± 0.02 mg/mL (for sample), and 2.05 ± 0.02 mg/mL (. Obtained results support the traditional use of these species for their beneficial health properties and suggest that investigated plant species could be potential sources of novel antiarthritic and anti-obesity agents.
Topics: Antioxidants; Methylene Chloride; Phytochemicals; Plant Extracts; Tropaeolum; Valerianaceae; Pancrelipase; Protein Denaturation; Anti-Obesity Agents
PubMed: 36615228
DOI: 10.3390/molecules28010032 -
Biophysical Journal Feb 2023Temperature is a useful system variable to gather kinetic and thermodynamic information from proteins. Usually, free energy and the associated entropic and enthalpic...
Temperature is a useful system variable to gather kinetic and thermodynamic information from proteins. Usually, free energy and the associated entropic and enthalpic contributions are obtained by quantifying the conformational equilibrium based on melting experiments performed in bulk conditions. Such experiments are suitable only for those small single-domain proteins whose side reactions of irreversible aggregation are unlikely to occur. Here, we avoid aggregation by pulling single-protein molecules in a thermo-regulated optical tweezers. Thus, we are able to explore the temperature dependence of the thermodynamic and kinetic parameters of MJ0366 from Methanocaldococcus jannaschii at the single-molecule level. By performing force-ramp experiments between 2°C and 40°C, we found that MJ0366 has a nonlinear dependence of free energy with temperature and a specific heat change of 2.3 ± 1.2 kcal/molK. These thermodynamic parameters are compatible with a two-state unfolding/refolding mechanism for MJ0366. However, the kinetics measured as a function of the temperature show a complex behavior, suggesting a three-state folding mechanism comprising a high-energy intermediate state. The combination of two perturbations, temperature and force, reveals a high-energy species in the folding mechanism of MJ0366 not detected in force-ramp experiments at constant temperature.
Topics: Temperature; Protein Folding; Optical Tweezers; Thermodynamics; Entropy; Kinetics; Protein Denaturation
PubMed: 36587240
DOI: 10.1016/j.bpj.2022.12.034 -
Scientific Reports Aug 2018Sweet proteins are an unexploited resource in the search for non-carbohydrate sweeteners mainly due to their low stability towards heating. Variants of the sweet protein...
Sweet proteins are an unexploited resource in the search for non-carbohydrate sweeteners mainly due to their low stability towards heating. Variants of the sweet protein monellin, with increased stability, were derived by an in vivo screening method based on the thermodynamic linkage between fragment complementation and protein stability. This approach depends on the correlation between mutational effects on the affinity between protein fragments and the stability of the intact protein. By linking the two fragments of monellin to the split GFP (green fluorescent protein) system, reconstitution of GFP was promoted and moderately fluorescent colonies were obtained. Two separate random libraries were produced for the monellin chains and the mutant clones were ranked based on fluorescence intensity. Mutants with increased affinity between the fragments, and subsequently increased stability, caused increased fluorescence intensity of split GFP. Single chain monellin variants of the top-ranked mutants for each chain, S76Y in the A-chain and W3C + R39G in the B-chain and all combinations thereof, were expressed and the increase in stability was verified by temperature denaturation studies using circular dichroism spectroscopy. Functionality studies showed that mutant S76Y has retained sweetness and has potential use within the food industry.
Topics: Amino Acid Sequence; Fluorescence; Green Fluorescent Proteins; Humans; Models, Molecular; Mutant Proteins; Mutation; Plant Proteins; Protein Denaturation; Protein Stability; Protein Structure, Secondary; Taste; Temperature; Terminology as Topic
PubMed: 30143736
DOI: 10.1038/s41598-018-31177-z -
Protein Science : a Publication of the... Dec 2022Protein folding and unfolding is a complex process, underscored by the many proteotoxic diseases associated with misfolded proteins. Mapping pathways from a native...
Protein folding and unfolding is a complex process, underscored by the many proteotoxic diseases associated with misfolded proteins. Mapping pathways from a native structure to an unfolded protein or vice versa, identifying the intermediates, and defining the role of sequence and structure en route remain outstanding problems in the field. It is even more challenging to capture the events at atomistic resolution. X-ray diffraction has so far been used to understand how urea interacts with and unfolds two stable globular proteins. Here, we present the case study on PSMD10 , a prototype for a moderately stable, non-globular repeat protein, long and rigid, with its termini located at either end. We define structural changes in the time dimension using low urea concentrations to arrive at the following conclusions. (a) Unfolding is rapidly initiated at the C-terminus, slowly at the N-terminus, and proceeds inwards from both ends. (b) C-terminus undergoes an α to 3 helix transition, representing the structure of a potential early unfolding intermediate before disorder sets in. (c) Distinct and progressive changes in the electrostatic landscape of PSMD10 were observed, indicative of conformational changes in the seemingly inflexible motif involved in protein-protein interaction. We believe this unique study will open up the field for better and bolder queries and increase the choice of model proteins for a better understanding of the challenging problems of protein folding, protein interactions, protein degradation, and diseases associated with misfolding.
Topics: Protein Denaturation; Urea; Ankyrin Repeat; Protein Folding; X-Ray Diffraction; Protein Conformation; Protein Unfolding
PubMed: 36382986
DOI: 10.1002/pro.4515 -
The Journal of Physical Chemistry. B Jul 2023Water is considered integral for the stabilization and function of proteins, which has recently attracted significant attention. However, the microscopic aspects of...
Water is considered integral for the stabilization and function of proteins, which has recently attracted significant attention. However, the microscopic aspects of water ranging up to the second hydration shell, including strongly and weakly bound water at the sub-nanometer scale, are not yet well understood. Here, we combined terahertz spectroscopy, thermal measurements, and infrared spectroscopy to clarify how the strongly and weakly bound hydration water changes upon protein denaturation. With denaturation, that is, the exposure of hydrophobic groups in water and entanglement of hydrophilic groups, the number of strongly bound hydration water decreased, while the number of weakly bound hydration water increased. Even though the constraint of water due to hydrophobic hydration is weak, it extends to the second hydration shell as it is caused by the strengthening of hydrogen bonds between water molecules, which is likely the key microscopic mechanism for the destabilization of the native state due to hydration.
Topics: Water; Proteins; Hydrophobic and Hydrophilic Interactions; Hydrogen Bonding; Protein Denaturation
PubMed: 37417885
DOI: 10.1021/acs.jpcb.3c02970 -
Chembiochem : a European Journal of... Jan 2018In unfolded proteins, peptide bonds involving Pro residues exist in equilibrium between the minor cis and major trans conformations. Folded proteins predominantly...
In unfolded proteins, peptide bonds involving Pro residues exist in equilibrium between the minor cis and major trans conformations. Folded proteins predominantly contain trans-Pro bonds, and slow cis-trans Pro isomerization in the unfolded state is often found to be a rate-limiting step in protein folding. Moreover, kinases and phosphatases that act upon Ser/Thr-Pro motifs exhibit preferential recognition of either the cis- or trans-Pro conformer. Here, NMR spectra obtained at both atmospheric and high pressures indicate that the population of cis-Pro falls well below previous estimates, an effect attributed to the use of short peptides with charged termini in most prior model studies. For the intrinsically disordered protein α-synuclein, cis-Pro populations at all of its five X-Pro bonds are less than 5 %, with only modest ionic strength dependence and no detectable effect of the previously demonstrated interaction between the N- and C-terminal halves of the protein. Comparison to small peptides with the same amino-acid sequence indicates that peptides, particularly those with unblocked, oppositely charged amino and carboxyl end groups, strongly overestimate the amount of cis-Pro.
Topics: Isomerism; Nuclear Magnetic Resonance, Biomolecular; Pressure; Proline; Protein Denaturation; alpha-Synuclein
PubMed: 29064600
DOI: 10.1002/cbic.201700548 -
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 -
International Journal of Molecular... Nov 2018The biological activity of proteins depends on their three-dimensional structure, known as the native state. The main force driving the correct folding mechanism is the... (Review)
Review
The biological activity of proteins depends on their three-dimensional structure, known as the native state. The main force driving the correct folding mechanism is the hydrophobic effect and when this folding kinetics is altered, aggregation phenomena intervene causing the occurrence of illnesses such as Alzheimer and Parkinson's diseases. The other important effect is performed by water molecules and by their ability to form a complex network of hydrogen bonds whose dynamics influence the mobility of protein amino acids. In this work, we review the recent results obtained by means of spectroscopic techniques, such as Fourier Transform Infrared (FTIR) and Nuclear Magnetic Resonance (NMR) spectroscopies, on hydrated lysozyme. In particular, we explore the Energy Landscape from the thermal region of configurational stability up to that of the irreversible denaturation. The importance of the coupling between the solute and the solvent will be highlighted as well as the different behaviors of hydrophilic and hydrophobic moieties of protein amino acid residues.
Topics: Animals; Humans; Hydrogen Bonding; Magnetic Resonance Spectroscopy; Protein Denaturation; Protein Folding; Spectroscopy, Fourier Transform Infrared
PubMed: 30513664
DOI: 10.3390/ijms19123825