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Chirality Sep 2022Protein-polymer conjugates are a blooming class of hybrid systems with high biomedical potential. Despite a plethora of papers on their biomedical properties, the...
Protein-polymer conjugates are a blooming class of hybrid systems with high biomedical potential. Despite a plethora of papers on their biomedical properties, the physical-chemical characterization of many protein-polymer conjugates is missing. Here, we evaluated the thermal stability of a set of fully-degradable polyphosphoester-protein conjugates by variable temperature circular dichroism, a common but powerful technique. We extensively describe their thermodynamic stability in different environments (in physiological buffer or in presence of chemical denaturants, e.g., acid or urea), highlighting the protective role of the polymer in preserving the protein from denaturation. For the first time, we propose a simple but effective protocol to achieve useful information on these systems in vitro, useful to screen new samples in their early stages.
Topics: Circular Dichroism; Myoglobin; Polymers; Protein Denaturation; Stereoisomerism; Thermodynamics
PubMed: 35713334
DOI: 10.1002/chir.23486 -
BioTechniques Oct 2012Protein stability can be monitored by many different techniques. However, these protocols are often lengthy, consume large amounts of protein, and require expensive and...
Protein stability can be monitored by many different techniques. However, these protocols are often lengthy, consume large amounts of protein, and require expensive and specialized instruments. Here we present a new protocol to analyze protein unfolding kinetics using a quantified real-time thermocycler. This technique enables the analysis of a wide range of denaturants (and their interactions with temperature change) on protein stability in a multi-well platform, where samples can be run in parallel under virtually identical conditions and with highly sensitive detection. Using this set-up, researchers can evaluate the half-maximal rate of protein denaturation (K(nd)), maximum rate of denaturation (D(max)), and the cooperativity of individual denaturants in protein unfolding (µ-coefficient). Both lysozyme and hexokinase are used as model proteins and urea as a model denaturant to illustrate this new method and the kinetics of protein unfolding that it provides. Overall, this method allows the researcher to explore a large number of denaturants, at either constant or variable temperatures, within the same assay, providing estimates of denaturation kinetics that have been previously inaccessible.
Topics: Differential Thermal Analysis; Kinetics; Protein Denaturation; Protein Stability
PubMed: 23046506
DOI: 10.2144/0000113922 -
Biophysical Journal Oct 2020Differential scanning calorimetry (DSC) indicated that PDZ3 undergoes a peculiar thermal denaturation, exhibiting two endothermic peaks because of the formation of...
Differential scanning calorimetry (DSC) indicated that PDZ3 undergoes a peculiar thermal denaturation, exhibiting two endothermic peaks because of the formation of reversible oligomers at high temperature (N↔I↔D). This contrasts sharply with the standard two-state denaturation model observed for small, globular proteins. We performed an alanine scanning analysis by individually mutating three hydrophobic residues at the crystallographic oligomeric interface (Phe340, Leu342, and Ile389) and one away from the interface (Leu349, as a control). DSC analysis indicated that PDZ3-F340A and PDZ3-L342A exhibited a single endothermic peak. Furthermore, PDZ3-L342A underwent a perfect two-state denaturation, as evidenced by the single endothermic peak and confirmed by detailed DSC analysis, including global fitting of data measured at different protein concentrations. Reversible oligomerization (RO) at high temperatures by small globular proteins is a rare event. Furthermore, our present study showing that a point mutation, L342A, designed based on the crystal structure inhibited RO is surprising because RO occurs at a high-temperature. Future studies will determine how and why mutations designed using crystal structures determined at ambient temperatures influence the formation of RO at high temperatures, and whether high-temperature ROs are related to the propensity of proteins to aggregate or precipitate at lower temperatures, which would provide a novel and unique way of controlling protein solubility and aggregation.
Topics: Calorimetry, Differential Scanning; Hot Temperature; Point Mutation; Protein Denaturation; Temperature; Thermodynamics
PubMed: 32961107
DOI: 10.1016/j.bpj.2020.08.023 -
Food Research International (Ottawa,... Sep 2021Milk protein concentrate-85 (MPC85) is a dairy ingredient which has a diverse range of applications in food products. The technofunctional properties of two MPC85...
Milk protein concentrate-85 (MPC85) is a dairy ingredient which has a diverse range of applications in food products. The technofunctional properties of two MPC85 samples having similar gross composition but different levels of native whey protein (WP), i.e., MPC85S1 and MPC85S2 with 16.6 and 6.0 g native WP/100 g protein, respectively, were compared. Rheometeric analysis showed that under an applied normal stress of 1.0-15.0 kPa, the compressibility, the air permeability and the cohesiveness of MPC85S2 was higher compared to MPC85S1. Differential scanning calorimetry showed that protein denaturation in MPC85S1 began at 63 °C while for MPC85S2 it began at 70 °C. The heat coagulation time (HCT at 140 °C) for 4.2% (w/v, on a protein basis) reconstituted MPC85S1 and MPC85S2 was 2.2 and 2.7 min, respectively. While a higher lightness for MPC85S1 was evidenced using colourimeter analysis, the colour stability on oven drying at 95 °C for MPC85S2 was higher than MPC85S1. The emulsion produced with MPC85S1 flocculated after 1 d and phase separation occurred after 14 d. In the case of MPC85S2, flocculation began after 4 d while phase separation was observed at 33 d. The viscosity of MPC85S2 (4.2% (w/v) protein) was higher than MPC85S1. This study showed differences between the flowability, viscosity, colour properties, thermal stability (in powder and in reconstituted format), emulsification and buffering capacity for MPC samples having two different levels of WP denaturation. The results demonstrated that the MPCs studied having two different levels of WP denaturation could be targeted for different functional applications. The minimal/maximum level of denaturation required to induce technofunctional property differences requires further study.
Topics: Desiccation; Hot Temperature; Milk Proteins; Protein Denaturation; Whey Proteins
PubMed: 34399548
DOI: 10.1016/j.foodres.2021.110576 -
Current Opinion in Structural Biology Apr 1999There have been a number of advances in atomic resolution simulations of biomolecules during the past few years. These have arisen partly from improvements to computer... (Review)
Review
There have been a number of advances in atomic resolution simulations of biomolecules during the past few years. These have arisen partly from improvements to computer power and partly from algorithmic improvements. There have also been advances in measuring time-dependent fluctuations in proteins using NMR spectroscopy, revealing the importance of fluctuations in the microsecond to millisecond time range. Progress has also been made in measuring how far the simulations are able to represent the accessible phase space that is available to the protein in its native state, in solution, at room temperature. Another area of development is the simulation of protein unfolding at atomic resolution.
Topics: Algorithms; Magnetic Resonance Spectroscopy; Models, Molecular; Protein Conformation; Protein Denaturation; Protein Folding; Proteins; Thermodynamics
PubMed: 10322213
DOI: 10.1016/S0959-440X(99)80022-0 -
Cell Stress & Chaperones Jul 2018Encysted embryos of Artemia are among the most stress-resistant eukaryotes partly due to the massive amount of a cysteine-rich protein termed artemin. High number of...
Encysted embryos of Artemia are among the most stress-resistant eukaryotes partly due to the massive amount of a cysteine-rich protein termed artemin. High number of cysteine residues in artemin and their intramolecular spatial positions motivated us to investigate the role of the cysteine residues in the chaperone-like activity of artemin. According to the result of Ellman's assay, there are nine free thiols (seven buried and two exposed) and one disulfide bond per monomer of artemin. Subsequent theoretical analysis of the predicted 3D structure of artemin confirmed the data obtained by the spectroscopic study. Native and reduced/modified forms of artemin were also compared with respect to their efficiency in chaperoning activity, tertiary structure, and stability. Since the alkylation and reduction of artemin diminished its chaperone activity, it appears that its chaperoning potential depends on the formation of intermolecular disulfide bond and the presence of cysteine residues. Comparative fluorescence studies on the structure and stability of the native and reduced protein revealed some differences between them. Due to the redox-dependent functional switching of artemin from the less to more active form, it can be finally suggested as a redox-dependent chaperone.
Topics: Animals; Arthropod Proteins; Cysteine; Disulfides; Guanidine; Iron-Binding Proteins; Molecular Chaperones; Molecular Dynamics Simulation; Oxidation-Reduction; Protein Denaturation; Protein Subunits; RNA-Binding Proteins; Recombinant Proteins; Structure-Activity Relationship; Thermodynamics
PubMed: 29429019
DOI: 10.1007/s12192-018-0880-7 -
Proceedings of the National Academy of... Nov 2008
Topics: Models, Molecular; Protein Denaturation; Proteins; Urea
PubMed: 18974225
DOI: 10.1073/pnas.0809224105 -
Proceedings of the National Academy of... Oct 2011To explain the large, opposite effects of urea and glycine betaine (GB) on stability of folded proteins and protein complexes, we quantify and interpret preferential... (Comparative Study)
Comparative Study
To explain the large, opposite effects of urea and glycine betaine (GB) on stability of folded proteins and protein complexes, we quantify and interpret preferential interactions of urea with 45 model compounds displaying protein functional groups and compare with a previous analysis of GB. This information is needed to use urea as a probe of coupled folding in protein processes and to tune molecular dynamics force fields. Preferential interactions between urea and model compounds relative to their interactions with water are determined by osmometry or solubility and dissected using a unique coarse-grained analysis to obtain interaction potentials quantifying the interaction of urea with each significant type of protein surface (aliphatic, aromatic hydrocarbon (C); polar and charged N and O). Microscopic local-bulk partition coefficients K(p) for the accumulation or exclusion of urea in the water of hydration of these surfaces relative to bulk water are obtained. K(p) values reveal that urea accumulates moderately at amide O and weakly at aliphatic C, whereas GB is excluded from both. These results provide both thermodynamic and molecular explanations for the opposite effects of urea and glycine betaine on protein stability, as well as deductions about strengths of amide NH--amide O and amide NH--amide N hydrogen bonds relative to hydrogen bonds to water. Interestingly, urea, like GB, is moderately accumulated at aromatic C surface. Urea m-values for protein folding and other protein processes are quantitatively interpreted and predicted using these urea interaction potentials or K(p) values.
Topics: Betaine; Binding Sites; Hydrogen Bonding; Models, Chemical; Molecular Dynamics Simulation; Protein Denaturation; Protein Folding; Protein Stability; Proteins; Urea
PubMed: 21930943
DOI: 10.1073/pnas.1109372108 -
PloS One 2012The C/EBP Homologous Protein (CHOP) is a nuclear protein that is integral to the unfolded protein response culminating from endoplasmic reticulum stress. Previously,...
The C/EBP Homologous Protein (CHOP) is a nuclear protein that is integral to the unfolded protein response culminating from endoplasmic reticulum stress. Previously, CHOP was shown to comprise extensive disordered regions and to self-associate in solution. In the current study, the intrinsically disordered nature of this protein was characterized further by comprehensive in silico analyses. Using circular dichroism, differential scanning calorimetry and nuclear magnetic resonance, we investigated the global conformation and secondary structure of CHOP and demonstrated, for the first time, that conformational changes in this protein can be induced by the free amino acid L-cysteine. Addition of L-cysteine caused a significant dose-dependent decrease in the protein helicity--dropping from 69.1% to 23.8% in the presence of 1 mM of L-cysteine--and a sequential transition to a more disordered state, unlike that caused by thermal denaturation. Furthermore, the presence of small amounts of free amino acid (80 µM, an 8:1 cysteine∶CHOP ratio) during CHOP thermal denaturation altered the molecular mechanism of its melting process, leading to a complex, multi-step transition. On the other hand, high levels (4 mM) of free L-cysteine seemed to cause a complete loss of rigid cooperatively melting structure. These results suggested a potential regulatory function of L-cysteine which may lead to changes in global conformation of CHOP in response to the cellular redox state and/or endoplasmic reticulum stress.
Topics: Circular Dichroism; Cysteine; Endoplasmic Reticulum Stress; Humans; Oxidation-Reduction; Protein Conformation; Protein Denaturation; Transcription Factor CHOP
PubMed: 22496840
DOI: 10.1371/journal.pone.0034680 -
Nucleic Acids Research Jul 2018Despite significant advances in the understanding of protein structure-function relationships, revealing protein folding pathways still poses a challenge due to a...
Despite significant advances in the understanding of protein structure-function relationships, revealing protein folding pathways still poses a challenge due to a limited number of relevant experimental tools. Widely-used experimental techniques, such as calorimetry or spectroscopy, critically depend on a proper data analysis. Currently, there are only separate data analysis tools available for each type of experiment with a limited model selection. To address this problem, we have developed the CalFitter web server to be a unified platform for comprehensive data fitting and analysis of protein thermal denaturation data. The server allows simultaneous global data fitting using any combination of input data types and offers 12 protein unfolding pathway models for selection, including irreversible transitions often missing from other tools. The data fitting produces optimal parameter values, their confidence intervals, and statistical information to define unfolding pathways. The server provides an interactive and easy-to-use interface that allows users to directly analyse input datasets and simulate modelled output based on the model parameters. CalFitter web server is available free at https://loschmidt.chemi.muni.cz/calfitter/.
Topics: Computational Biology; Internet; Models, Molecular; Protein Denaturation; Protein Folding; Protein Unfolding; Software
PubMed: 29762722
DOI: 10.1093/nar/gky358