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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 -
Ultrasonics Sonochemistry Jul 2019The influence of ultrasonics combined with microwave thawing (UMT) and ultrasonics combined with far-infrared thawing (UIT) on the water migration and protein...
The influence of ultrasonics combined with microwave thawing (UMT) and ultrasonics combined with far-infrared thawing (UIT) on the water migration and protein denaturation of red drum were studied. Five treatments were used: ultrasonics thawing (UT), microwave thawing (MT), far-infrared thawing (IT), UIT and UMT were used for thawing red drum filets. Moisture migration and protein aggregation were studied using nuclear magnetic resonance (NMR) and particle size, respectively. Raman spectra and fluorescence spectra were used to study the secondary and tertiary structure of protein. SEM was done to observe the fiber microstructure. The results showed that UMT and UIT can maintained protein stability more than other thawing methods and retained the fiber structure of the muscle. Besides, immobilized water in fiber bundles network also had no significant changes compared with fresh samples. Thus, ultrasonics combined with far infrared or microwave thawing were used to decrease protein denaturation and water migration during the thawing of Red drum fillets.
Topics: Animals; Fish Proteins; Infrared Rays; Microwaves; Perciformes; Protein Aggregates; Protein Denaturation; Water
PubMed: 31084796
DOI: 10.1016/j.ultsonch.2019.03.017 -
European Journal of Pharmaceutics and... Nov 2023Assessment of cold stability is essential for manufacture and commercialization of biotherapeutics. Storage stability is often estimated by measuring accelerated rates...
Assessment of cold stability is essential for manufacture and commercialization of biotherapeutics. Storage stability is often estimated by measuring accelerated rates at elevated temperature and using mathematical models (as the Arrhenius equation). Although, this strategy often leads to an underestimation of protein aggregation during storage. In this work, we measured the aggregation rates of two antibodies in a broad temperature range (from 60 °C to -25 °C), using an isochoric cooling method to prevent freezing of the formulations below 0 °C. Both antibodies evidenced increasing aggregation rates when approaching extreme temperatures, because of hot and cold denaturation. This behavior was modelled using Arrhenius and Gibbs-Helmholtz equations, which enabled to deconvolute the contribution of unfolding from the protein association kinetics. This approach made possible to model the aggregation rates at refrigeration temperature (5 °C) in a relatively short timeframe (1-2 weeks) and using standard characterization techniques (SEC-HPLC and DLS).
Topics: Protein Stability; Cold Temperature; Temperature; Freezing; Antibodies; Protein Denaturation
PubMed: 37832611
DOI: 10.1016/j.ejpb.2023.10.009 -
The Journal of Biological Chemistry Mar 1990The results of a thermodynamic calculation of the excess heat capacity that is based on experimental observations and that incorporates the effects of ligand binding on...
The results of a thermodynamic calculation of the excess heat capacity that is based on experimental observations and that incorporates the effects of ligand binding on the two-state, thermal denaturation of a protein are presented. For a protein with a single-binding site on the native species and at subsaturating concentrations of ligand, bimodal or unimodal thermograms were computed merely by assuming a larger or smaller ligand association constant, respectively. The calculated thermograms for this simplified case show the salient features of those observed by differential scanning calorimetry for defatted human albumin monomer in the absence and presence of three ligands for which the protein has higher, intermediate, and lower affinity (Shrake, A., and Ross, P. D. (1988) J. Biol. Chem. 263, 15392-15399). The computation demonstrates that biphasic unfolding can result from a significant increase in the free energy of denaturation (and the transition temperature) during the course of unfolding due to a substantial increase in free ligand concentration caused by the release of bound ligand by denaturing protein. Such ligand-induced biphasic denaturation does not relate to macromolecular substructure but derives from a perturbation, during unfolding, of the ligand binding equilibrium, which is coupled to the equilibrium between the folded and unfolded protein species. Thus, this bimodality is not limited to thermally induced unfolding but is operative independent of the means used to effect denaturation and therefore must be considered when studying any macromolecular folding/unfolding reaction in the presence of ligand.
Topics: Kinetics; Ligands; Mathematics; Protein Denaturation; Thermodynamics
PubMed: 2318882
DOI: No ID Found -
Proceedings of the National Academy of... May 1988Denaturation of staphylococcal nuclease was studied in a temperature range from -7 to 70 degrees C by scanning microcalorimetry and spectropolarimetry. It was found that...
Denaturation of staphylococcal nuclease was studied in a temperature range from -7 to 70 degrees C by scanning microcalorimetry and spectropolarimetry. It was found that the native protein is maximally stable at about 20 degrees C and is denatured upon heating and cooling from this temperature. The heat and cold denaturation processes are approximated rather well by a two-state transition showing that the molecule is composed of a single cooperative system. The main difference between these two processes is in the sign of the enthalpy and entropy of denaturation: whereas the heat denaturation proceeds with increases in the enthalpy and entropy, the cold denaturation proceeds with decreases in both quantities. The inversion of the enthalpy sign occurs at about 15 degrees C in an acetate buffer, but this temperature can be raised by addition of urea to the solvent.
Topics: Calorimetry; Cold Temperature; Micrococcal Nuclease; Protein Denaturation
PubMed: 3368446
DOI: 10.1073/pnas.85.10.3343 -
Proceedings of the National Academy of... Oct 2013Protein folding has been extensively studied, but many questions remain regarding the mechanism. Characterizing early unstable intermediates and the high-free-energy...
Protein folding has been extensively studied, but many questions remain regarding the mechanism. Characterizing early unstable intermediates and the high-free-energy transition state (TS) will help answer some of these. Here, we use effects of denaturants (urea, guanidinium chloride) and temperature on folding and unfolding rate constants and the overall equilibrium constant as probes of surface area changes in protein folding. We interpret denaturant kinetic m-values and activation heat capacity changes for 13 proteins to determine amounts of hydrocarbon and amide surface buried in folding to and from TS, and for complete folding. Predicted accessible surface area changes for complete folding agree in most cases with structurally determined values. We find that TS is advanced (50-90% of overall surface burial) and that the surface buried is disproportionately amide, demonstrating extensive formation of secondary structure in early intermediates. Models of possible pre-TS intermediates with all elements of the native secondary structure, created for several of these proteins, bury less amide and hydrocarbon surface than predicted for TS. Therefore, we propose that TS generally has both the native secondary structure and sufficient organization of other regions of the backbone to nucleate subsequent (post-TS) formation of tertiary interactions. The approach developed here provides proof of concept for the use of denaturants and other solutes as probes of amount and composition of the surface buried in coupled folding and other large conformational changes in TS and intermediates in protein processes.
Topics: Models, Chemical; Protein Denaturation; Protein Folding; Proteins
PubMed: 24043778
DOI: 10.1073/pnas.1311948110 -
Journal of Synchrotron Radiation Mar 2009Investigation of radiation damage in protein crystals has progressed in several directions over the past couple of years. There have been improvements in the basic... (Review)
Review
Investigation of radiation damage in protein crystals has progressed in several directions over the past couple of years. There have been improvements in the basic procedures such as calibration of the incident X-ray intensity and calculation of the dose likely to be deposited in a crystal of known size and composition with this intensity. There has been increased emphasis on using additional techniques such as optical, Raman or X-ray spectroscopy to complement X-ray diffraction. Apparent discrepancies between the results of different techniques can be explained by the fact that they are sensitive to different length scales or to changes in the electronic state rather than to movement of atoms. Investigations have been carried out at room temperature as well as cryo-temperatures and, in both cases, with the introduction of potential scavenger molecules. These and other studies are leading to an overall description of the changes which can occur when a protein crystal is irradiated with X-rays at both cryo- and room temperatures. Results from crystallographic and spectroscopic radiation-damage experiments can be reconciled with other studies in the field of radiation physics and chemistry.
Topics: Crystallization; Crystallography, X-Ray; Protein Conformation; Protein Denaturation; Proteins; Radiation Dosage; Specimen Handling; X-Rays
PubMed: 19240324
DOI: 10.1107/S0909049509005238 -
Biophysical Chemistry Dec 2017Simulations of protein thermodynamics are generally difficult to perform and provide limited information. It is desirable to increase the degree of detail provided by...
Simulations of protein thermodynamics are generally difficult to perform and provide limited information. It is desirable to increase the degree of detail provided by simulation and thereby the potential insight into the thermodynamic properties of proteins. In this study, we outline how to analyze simulation trajectories to decompose conformation-specific, parameter free, thermodynamically defined protein volumes into residue-based contributions. The total volumes are obtained using established methods from Fluctuation Solution Theory, while the volume decomposition is new and is performed using a simple proximity method. Native and fully extended ubiquitin are used as the test conformations. Changes in the protein volumes are then followed as a function of pressure, allowing for conformation-specific protein compressibility values to also be obtained. Residue volume and compressibility values indicate significant contributions to protein denaturation thermodynamics from nonpolar and coil residues, together with a general negative compressibility exhibited by acidic residues.
Topics: Molecular Dynamics Simulation; Pressure; Protein Denaturation; Protein Stability; Thermodynamics; Ubiquitin
PubMed: 28576277
DOI: 10.1016/j.bpc.2017.04.006 -
International Journal of Biological... Sep 2019Effects of a beetle antifreeze proteins (AFP) from Dendroides canadensis (DAFP-1) on a model freeze-labile enzyme, lactate dehydrogenase (LDH), were investigated under...
Effects of a beetle antifreeze proteins (AFP) from Dendroides canadensis (DAFP-1) on a model freeze-labile enzyme, lactate dehydrogenase (LDH), were investigated under freezing and thawing conditions. The presence of DAFP-1 can effectively protect the enzymatic activity of LDH upon repeated freezing and thawing and the protective role of DAFP-1 is more significant than that of bovine serum albumin (BSA), a common protectant for freeze-labile proteins. The results of circular dichroism (CD) spectroscopy suggest that the presence of DAFP-1 provides protection to the denaturation of LDH under freezing and thawing. The molecular dynamics (MD) simulation of DAFP-1 and LDH suggests that DAFP-1 interacts with LDH using its ice-binding surface (IBS) and mainly through its arginine residues. A mutant of DAFP-1, where all the arginine residues were replaced by alanine residues, lost its effect in protecting LDH under freezing and thawing. The results demonstrated that DAFP-1 is an effective protectant for a freeze-labile protein under freezing and thawing and the arginine residues in DAFP-1 are important for its protective role. By correlating the protective effect of an AFP with its structure, new insights in the identification and development of effective protectants for freeze-labile proteins were provided.
Topics: Animals; Antifreeze Proteins; Coleoptera; Freezing; Insect Proteins; L-Lactate Dehydrogenase; Molecular Dynamics Simulation; Protein Conformation; Protein Denaturation
PubMed: 31226372
DOI: 10.1016/j.ijbiomac.2019.06.067 -
Journal of Oleo Science 2018Protein denaturants play an important role in medical and biological research, and development of new denaturants is widely explored to study aging and various diseases....
Protein denaturants play an important role in medical and biological research, and development of new denaturants is widely explored to study aging and various diseases. In this research, we treated lysozyme, a model protein, with photocatalysts of ground Rh-doped SrTiO (g-STO:Rh) and ground Rh-Sb-co-doped SrTiO (g-STO:Rh/Sb) under visible light irradiation to explore the potential of those photocatalysts as denaturants. SDS-PAGE showed that photocatalysis with g-STO:Rh induced the fragmentation of lysozyme into unidentifiable decomposition products. BCA and Bradford protein assays indicated that the peptide bonds and basic, aromatic and N-terminal amino acid residues in lysozyme were denaturated by g-STO:Rh photocatalysis. The denaturation of those amino acids, as quantified by the decreased solubility of lysozyme, was estimated to be more severe by Bradford protein assay than by BCA protein assay. Circular dichroism (CD) spectra of lysozyme revealed that the secondary structure was denatured by g-STO:Rh photocatalysis, indicating that g-STO:Rh photocatalysis is especially effective against the amino acid residues that form the secondary structure via hydrogen bonds. Furthermore, the lytic activity of lysozyme was reduced by g-STO:Rh photocatalysis, owing to denaturation of the enzyme. The visible-light-responsive photocatalyst of g-STO:Rh/Sb accelerates the oxidation reaction and has stronger oxidizing power than g-STO:Rh. Lysozyme was denatured more quickly by g-STO:Rh/Sb photocatalysis than by g-STO:Rh according to analysis by SDS-PAGE, CD spectroscopy, BCA and Bradford protein assays, and lytic activity. These results suggest that higher photocatalytic activity induces more significant denaturation of lysozyme, implying that the main factor of photocatalytic denaturation of lysozyme is oxidation. It should be noted that, as far as we know, this is the first report for denaturation of protein using visible-light-responsive photocatalyst.
Topics: Antimony; Catalysis; Light; Muramidase; Oxidation-Reduction; Oxides; Protein Denaturation; Protein Structure, Secondary; Rhodium; Strontium; Titanium
PubMed: 30504623
DOI: 10.5650/jos.ess18155