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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 -
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 -
Scientific Reports Dec 2021SARS-CoV-2, the virus that causes COVID-19, is still a widespread threat to society. The spike protein of this virus facilitates viral entry into the host cell. Here,...
SARS-CoV-2, the virus that causes COVID-19, is still a widespread threat to society. The spike protein of this virus facilitates viral entry into the host cell. Here, the denaturation of the S1 subunit of this spike protein by 2.45 GHz electromagnetic radiation was studied quantitatively. The study only pertains to the pure electromagnetic effects by eliminating the bulk heating effect of the microwave radiation in an innovative setup that is capable of controlling the temperature of the sample at any desired intensity of the electromagnetic field. This study was performed at the internal human body temperature, 37 °C, for a relatively short amount of time under a high-power electromagnetic field. The results showed that irradiating the protein with a 700 W, 2.45 GHz electromagnetic field for 2 min can denature the protein to around 95%. In comparison, this is comparable to thermal denaturation at 75 °C for 40 min. Electromagnetic denaturation of the proteins of the virus may open doors to potential therapeutic or sanitation applications.
Topics: Microwaves; Protein Denaturation; SARS-CoV-2; Spike Glycoprotein, Coronavirus; Temperature
PubMed: 34862423
DOI: 10.1038/s41598-021-02753-7