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Journal of the Royal College of... Oct 1989
Review
Topics: Aging; Free Radicals; Humans; Inflammation; Lipid Peroxidation; Neoplasms; Protein Denaturation; Reperfusion Injury
PubMed: 2685266
DOI: No ID Found -
Analytical Biochemistry Mar 2015The determination of accurate binding affinities is critical in drug discovery and development. Several techniques are available for characterizing the binding of small...
The determination of accurate binding affinities is critical in drug discovery and development. Several techniques are available for characterizing the binding of small molecules to soluble proteins. The situation is different for integral membrane proteins. Isothermal chemical denaturation has been shown to be a valuable biophysical method to determine, in a direct and label-free fashion, the binding of ligands to soluble proteins. In this study, the application of isothermal chemical denaturation was applied to an integral membrane protein, the A2a G-protein coupled receptor. Binding affinities for a set of 19 small molecule agonists/antagonists of the A2a receptor were determined and found to be in agreement with data from surface plasmon resonance and radioligand binding assays previously reported in the literature. Therefore, isothermal chemical denaturation expands the available toolkit of biophysical techniques to characterize and study ligand binding to integral membrane proteins, specifically G-protein coupled receptors in vitro.
Topics: Adenosine A2 Receptor Agonists; Adenosine A2 Receptor Antagonists; Biophysics; Guanidine; Ligands; Protein Binding; Protein Denaturation; Receptor, Adenosine A2A; Temperature
PubMed: 25481736
DOI: 10.1016/j.ab.2014.11.019 -
Biophysical Chemistry Feb 2018Knowledge of protein stability is of utmost importance in various fields of biotechnology. Protein stability can be assessed in solution by increasing the concentration...
Knowledge of protein stability is of utmost importance in various fields of biotechnology. Protein stability can be assessed in solution by increasing the concentration of denaturant and recording the structural changes with spectroscopic or thermodynamic methods. The standard interpretation of the experimental data is to assume a 2-state equilibrium between completely folded and completely unfolded protein molecules. Here we propose a cooperative model based on the statistical-mechanical Zimm-Bragg theory. In this model protein unfolding is driven by the weak binding of a rather small number of denaturant molecules, inducing the cooperative unfolding with multiple dynamic intermediates. The modified Zimm-Bragg theory is applied to published thermodynamic and spectroscopic data leading to the following conclusions. (i) The binding constant K is correlated with the midpoint concentration, c, of the unfolding reaction according to c≅1/K. The better the binding of denaturant the lower is the concentration to achieve unfolding. (ii) The binding constant K agrees with direct thermodynamic measurements. A rather small number of bound denaturants suffices to induce the cooperative unfolding of the whole protein. (iii) Chemical unfolding occurs in the concentration range Δc=c-c. The theory predicts the unfolding energy per amino acid residue as g=RTK(c-c). The Gibbs free energy of an osmotic gradient of the same size is ΔG=-RTln(c/c). In all examples investigated ΔG exactly balances the unfolding energy g. The total unfolding energy is thus close to zero. (iv) Protein cooperativity in chemical unfolding is rather low with cooperativity parameters σ≥3x10. As a consequence, the theory predicts a dynamic mixture of conformations during the unfolding reaction. The probabilities of individual conformations are easily accessible via the partition function Z(c,σ).
Topics: Bacterial Proteins; Chymotrypsin; Hydrochloric Acid; Models, Statistical; Muramidase; Phosphoenolpyruvate Sugar Phosphotransferase System; Protein Denaturation; Protein Unfolding; Thermodynamics; Urea
PubMed: 29232602
DOI: 10.1016/j.bpc.2017.12.001 -
Biomolecular NMR Assignments Apr 2018Brain derived neurotrophic factor (BDNF) is a member of the neurotrophin family of proteins which plays a central role in neuronal survival, growth, plasticity and...
Brain derived neurotrophic factor (BDNF) is a member of the neurotrophin family of proteins which plays a central role in neuronal survival, growth, plasticity and memory. A single Val66Met variant has been identified in the prodomain of human BDNF that is associated with anxiety, depression and memory disorders. The structural differences within the full-length prodomain Val66 and Met66 isoforms could shed light on the mechanism of action of the Met66 and its impact on the development of neuropsychiatric-associated disorders. In the present study, we report the backbone H, C, and N NMR assignments of both full-length Val66 and Met66 prodomains in the presence of 2 M urea. These conditions were utilized to suppress residual structure and aid subsequent native state structural investigations aimed at mapping and identifying variant-dependent conformational differences under native-state conditions.
Topics: Brain-Derived Neurotrophic Factor; Humans; Nuclear Magnetic Resonance, Biomolecular; Protein Denaturation; Protein Domains; Urea
PubMed: 28933046
DOI: 10.1007/s12104-017-9777-0 -
Protein Science : a Publication of the... Apr 2001Muscle of amphioxus contains large amounts of a four EF-hand Ca2+-binding protein, CaVP, and its target, CaVPT. To study the domain structure of CaVP and assess the...
Muscle of amphioxus contains large amounts of a four EF-hand Ca2+-binding protein, CaVP, and its target, CaVPT. To study the domain structure of CaVP and assess the structurally important determinants for its interaction with CaVPT, we expressed CaVP and its amino (N-CaVP) and carboxy-terminal halves (C-CaVP). The interactive properties of recombinant and wild-type CaVP are very similar, despite three post-translational modifications in the wild-type protein. N-CaVP does not bind Ca2+, shows a well-formed hydrophobic core, and melts at 44 degrees C. C-CaVP binds two Ca2+ with intrinsic dissociation constants of 0.22 and 140 microM (i.e., very similar to the entire CaVP). The metal-free domain in CaVP and C-CaVP shows no distinct melting transition, whereas its 1Ca2+ and 2Ca2+) forms melt in the 111 degrees -123 degrees C range, suggesting that C-CaVP and the carboxy- domain of CaVP are natively unfolded in the metal-free state and progressively gain structure upon binding of 1Ca2+ and 2Ca2+. Thermal denaturation studies provide evidence for interdomain interaction: the apo, 1Ca2+ and 2Ca2+ states of the carboxy-domain destabilize to different degrees the amino-domain. Only C-CaVP forms a Ca2+-dependent 1:1 complex with CaVPT. Our results suggest that the carboxy-terminal domain of CaVP interacts with CaVPT and that the amino-terminal lobe modulates this interaction.
Topics: Amino Acid Sequence; Animals; Calcium-Binding Proteins; Circular Dichroism; EF Hand Motifs; Escherichia coli; Muscle Proteins; Protein Conformation; Protein Denaturation; Protein Folding; Protein Structure, Tertiary; Recombinant Proteins; Thermodynamics
PubMed: 11274468
DOI: 10.1110/ps.40601 -
Journal of the American Chemical Society Oct 2009The study of protein folding requires a method to drive unfolding, which is typically accomplished by altering solution conditions to favor the denatured state. This has...
The study of protein folding requires a method to drive unfolding, which is typically accomplished by altering solution conditions to favor the denatured state. This has the undesirable consequence that the molecular forces responsible for configuring the polypeptide chain are also changed. It would therefore be useful to develop methods that can drive unfolding without the need for destabilizing solvent conditions. Here we introduce a new method to accomplish this goal, which we call steric trapping. In the steric trap method, the target protein is labeled with two biotin tags placed close in space so that both biotin tags can only be bound by streptavidin when the protein unfolds. Thus, binding of the second streptavidin is energetically coupled to unfolding of the target protein. Testing the method on a model protein, dihydrofolate reductase (DHFR), we find that streptavidin binding can drive unfolding and that the apparent binding affinity reports on changes in DHFR stability. Finally, by employing the slow off-rate of wild-type streptavidin, we find that DHFR can be locked in the unfolded state. The steric trap method provides a simple method for studying aspects of protein folding and stability in native solvent conditions, could be used to specifically unfold selected domains, and could be applicable to membrane proteins.
Topics: Animals; Biotinylation; Mice; Models, Molecular; Protein Conformation; Protein Denaturation; Protein Folding; Streptavidin; Tetrahydrofolate Dehydrogenase
PubMed: 19739627
DOI: 10.1021/ja905725n -
Protein Science : a Publication of the... Jul 2003Standard methods for measuring free energy of protein unfolding by chemical denaturation require complete folding at low concentrations of denaturant so that a native...
Standard methods for measuring free energy of protein unfolding by chemical denaturation require complete folding at low concentrations of denaturant so that a native baseline can be observed. Alternatively, proteins that are completely unfolded in the absence of denaturant can be folded by addition of the osmolyte trimethylamine N-oxide (TMAO), and the unfolding free energy can then be calculated through analysis of the refolding transition. However, neither chemical denaturation nor osmolyte-induced refolding alone is sufficient to yield accurate thermodynamic unfolding parameters for partly folded proteins, because neither method produces both native and denatured baselines in a single transition. Here we combine urea denaturation and TMAO stabilization as a means to bring about baseline-resolved structural transitions in partly folded proteins. For Barnase and the Notch ankyrin domain, which both show two-state equilibrium unfolding, we found that DeltaG degrees for unfolding depends linearly on TMAO concentration, and that the sensitivity of DeltaG degrees to urea (the m-value) is TMAO independent. This second observation confirms that urea and TMAO exert independent effects on stability over the range of cosolvent concentrations required to bring about baseline-resolved structural transitions. Thermodynamic parameters calculated using a global fit that assumes additive, linear dependence of DeltaG degrees on each cosolvent are similar to those obtained by standard urea-induced unfolding in the absence of TMAO. Finally, we demonstrate the applicability of this method to measurement of the free energy of unfolding of a partly folded protein, a fragment of the full-length Notch ankyrin domain.
Topics: Methylamines; Osmolar Concentration; Oxidants; Protein Denaturation; Protein Folding; Proteins; Solvents; Thermodynamics; Urea
PubMed: 12824497
DOI: 10.1110/ps.0372903 -
Analytical Chemistry Nov 2019Assessing the physical stability of proteins is one of the most important challenges in the development, manufacture, and formulation of biotherapeutics. Here, we...
Assessing the physical stability of proteins is one of the most important challenges in the development, manufacture, and formulation of biotherapeutics. Here, we describe a method for combining and automating circular dichroism and intrinsic protein fluorescence spectroscopy. By robotically injecting samples from a 96-well plate into an optically compliant capillary flow cell, complementary information about the secondary and tertiary structural state of a protein can be collected in an unattended manner from considerably reduced volumes of sample compared to conventional techniques. We demonstrate the accuracy and reproducibility of this method. Furthermore, we show how structural screening can be used to monitor unfolding of proteins in two case studies using (i) a chaotropic denaturant (urea) and (ii) low-pH buffers used for monoclonal antibody (mAb) purification during Protein A chromatography.
Topics: Automation; Circular Dichroism; Hydrogen-Ion Concentration; Protein Conformation; Protein Denaturation; Protein Folding; Protein Structure, Secondary; Protein Structure, Tertiary; Reproducibility of Results; Spectrometry, Fluorescence; Urea
PubMed: 31584804
DOI: 10.1021/acs.analchem.9b03259 -
Proteins Sep 2010Proteins aggregate in response to various stresses including changes in solvent conditions. Addition of alcohols has been recently shown to induce aggregation of...
Proteins aggregate in response to various stresses including changes in solvent conditions. Addition of alcohols has been recently shown to induce aggregation of disease-related as well as nondisease-related proteins. Here we probed the biophysical mechanisms underlying alcohol-induced protein aggregation, in particular the role of partial protein unfolding in aggregation. We have studied aggregation mechanisms due to benzyl alcohol which is used in numerous biochemical and biotechnological applications. We chose cytochrome c as a model protein, for the reason that various optical and structural probes are available to monitor its global and partial unfolding reactions. Benzyl alcohol induced the aggregation of cytochrome c in isothermal conditions and decreased the temperature at which the protein aggregates. However, benzyl alcohol did not perturb the overall native conformation of cytochrome c. Instead, it caused partial unfolding of a local protein region around the methionine residue at position 80. Site-specific optical probes, two-dimensional NMR titrations, and hydrogen exchange all support this conclusion. The protein aggregation temperature varied linearly with the melting temperature of the Met80 region. Stabilizing the Met80 region by heme iron reduction drastically decreased protein aggregation, which confirmed that the local unfolding of this region causes protein aggregation. These results indicate that a possible mechanism by which alcohols induce protein aggregation is through partial rather than complete unfolding of native proteins.
Topics: Anesthetics, Local; Animals; Benzyl Alcohol; Cytochromes c; Horses; Models, Molecular; Protein Conformation; Protein Denaturation; Protein Folding; Protein Multimerization; Solvents; Temperature
PubMed: 20597088
DOI: 10.1002/prot.22778 -
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