Chapter 8. Protein Thermal Denaturation Measurements via a Fluorescent Dye

Cimmperman, P.; Matulis, Daumantas

doi:10.1039/9781849732666-00247

Cited by 36 publications

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“…It meant that the dye was in part initially fixed on hydrophobic domains which could be located either on the capsid surface or/and inside the viral particle thus implying possible dye penetration. On the DSF profile, the decrease in fluorescence between 20 and 60°C for heated phages could be due to probe quenching by water (Cimmperman and Matulis 2011). The T m value of 66-67°C was still observed on the DSF profile after a 60°C treatment.…”

Section: Effect Of Heat On the Hydrophobicity Of Ms2 Phage Particlesmentioning

confidence: 65%

The Effect of Heat on the Physicochemical Properties of Bacteriophage MS2

et al. 2016

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The differences in physicochemical characteristics between infectious and non-infectious viral particles are poorly known. Even for heat, which is known as one of the most efficient treatments to inactivate enteric viruses, the global inactivation mechanisms have not been described yet. Such knowledge would help distinguish between both types of particles and therefore clarify the interpretation of the presence of viral genomes in food after heat treatment. In this study, we examined in particular the differences in electrostatic charge and hydrophobicity between the two particle types. MS2 phage, a common surrogate for enteric viruses, was used as a model virus. The heat-induced inactivation process of the infectious phages caused hydrophobic domains to be transiently exposed and their charge to become less negative. The particles also became progressively permeable to small molecules such as SYPRO Orange dye. The presence of non-infectious phage particles in which the genome was not accessible to RNases has been clearly demonstrated. These observations were done for MS2 phages exposed to a temperature of 60 °C. When exposed to a temperature higher than their critical temperature (72 °C), the particles were disrupted and the genome became available for RNases. At lower temperatures, 60 °C in this study, the transient expression of hydrophobic domains of remaining infectious phages appeared as an interesting parameter for improving their specific detection.

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Section: Effect Of Heat On the Hydrophobicity Of Ms2 Phage Particlesmentioning

confidence: 65%

The Effect of Heat on the Physicochemical Properties of Bacteriophage MS2

et al. 2016

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“…( b ) The dose response curves as a function of added concentration of EZA (◊) or TFMSA (■). Datapoints are obtained from fluorescence melting curves such as in panel (a), while the lines are fitted according to [27-29]. The addition of EZA shifted the T m s more than TFMSA at the same concentration.…”

Section: Resultsmentioning

confidence: 99%

“…The pH dependence of the K b was determined in the universal buffer containing 50 mM sodium phosphate, 50 mM sodium acetate, and 25 mM sodium borate. The fluorescence and ligand dosing curves were fit as previously described [27,28]. TSA experiments were repeated at least three times.…”

Section: Methodsmentioning

confidence: 99%

“…However, it is not appropriate for determining weak (millimolar) or very tight dissociation constants (subnanomolar K d s require displacement ITC [21]) and consumes extensive amounts of protein and time. In contrast, TSA is a rapid screening method used in pharmaceutical industry for the identification of binders and requires lower amounts of protein [22-27]. The method is based on the protein melting temperature ( T m ) shift that occurs upon ligand binding.…”

Section: Introductionmentioning

confidence: 99%

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Intrinsic thermodynamics of ethoxzolamide inhibitor binding to human carbonic anhydrase XIII

Baranauskienė

Matulis

2012

BMC Biophys

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BackgroundHuman carbonic anhydrases (CAs) play crucial role in various physiological processes including carbon dioxide and hydrocarbon transport, acid homeostasis, biosynthetic reactions, and various pathological processes, especially tumor progression. Therefore, CAs are interesting targets for pharmaceutical research. The structure-activity relationships (SAR) of designed inhibitors require detailed thermodynamic and structural characterization of the binding reaction. Unfortunately, most publications list only the observed thermodynamic parameters that are significantly different from the intrinsic parameters. However, only intrinsic parameters could be used in the rational design and SAR of the novel compounds.ResultsIntrinsic binding parameters for several inhibitors, including ethoxzolamide, trifluoromethanesulfonamide, and acetazolamide, binding to recombinant human CA XIII isozyme were determined. The parameters were the intrinsic Gibbs free energy, enthalpy, entropy, and the heat capacity. They were determined by titration calorimetry and thermal shift assay in a wide pH and temperature range to dissect all linked protonation reaction contributions.ConclusionsPrecise determination of the inhibitor binding thermodynamics enabled correct intrinsic affinity and enthalpy ranking of the compounds and provided the means for SAR analysis of other rationally designed CA inhibitors.

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“…In this case, it is important to know to what extent the crystalline state affects the binding of the ligand and in particular its affinity. For instance, it is known that ligands often increase the order of the target protein 5. This may have an effect on packing contacts.…”

Section: Introductionmentioning

confidence: 99%

Crystal packing modifies ligand binding affinity: The case of aldose reductase

et al. 2012

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The relationship between the structures of protein–ligand complexes existing in the crystal and in solution, essential in the case of fragment-based screening by X-ray crystallography (FBS-X), has been often an object of controversy. To address this question, simultaneous co-crystallization and soaking of two inhibitors with different ratios, Fidarestat (FID; Kd = 6.5 nM) and IDD594 (594; Kd = 61 nM), which bind to h-aldose reductase (AR), have been performed. The subatomic resolution of the crystal structures allows the differentiation of both inhibitors, even when the structures are almost superposed. We have determined the occupation ratio in solution by mass spectrometry (MS) Occ(FID)/Occ(594) = 2.7 and by X-ray crystallography Occ(FID)/Occ(594) = 0.6. The occupancies in the crystal and in solution differ 4.6 times, implying that ligand binding potency is influenced by crystal contacts. A structural analysis shows that the Loop A (residues 122–130), which is exposed to the solvent, is flexible in solution, and is involved in packing contacts within the crystal. Furthermore, inhibitor 594 contacts the base of Loop A, stabilizing it, while inhibitor FID does not. This is shown by the difference in B-factors of the Loop A between the AR–594 and AR–FID complexes. A stable loop diminishes the entropic energy barrier to binding, favoring 594 versus FID. Therefore, the effect of the crystal environment should be taken into consideration in the X-ray diffraction analysis of ligand binding to proteins. This conclusion highlights the need for additional methodologies in the case of FBS-X to validate this powerful screening technique, which is widely used.

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Chapter 8. Protein Thermal Denaturation Measurements via a Fluorescent Dye

Cited by 36 publications

References 0 publications

The Effect of Heat on the Physicochemical Properties of Bacteriophage MS2

The Effect of Heat on the Physicochemical Properties of Bacteriophage MS2

Intrinsic thermodynamics of ethoxzolamide inhibitor binding to human carbonic anhydrase XIII

Crystal packing modifies ligand binding affinity: The case of aldose reductase

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