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A thermal shift assay quantifies the change in thermal denaturation temperature of a protein under varying conditions. The differing conditions that can be examined are very diverse, e.g. pH, salts, additives, drugs, drug leads, oxidation/reduction, or mutations. The binding of low molecular weight ligands can increase the thermal stability of a protein, as described by Koshland (1958) and Linderstrom-Lang and Schellman (1959).〔Linderstrøm-Lang, K., and Schellman, J. A. (1959). "Protein structure and enzyme activity". ''The Enzymes.'' 1(2) 443-510.〕 Almost half of enzymes require a metal ion co-factor. Thermostable proteins are often more useful than their non-thermostable counterparts, e.g. DNA polymerase in the polymerase chain reaction, so protein engineering often includes adding mutations to increase thermal stability. Protein crystallisation is more successful for proteins with a higher melting point and adding buffer components that stabilise proteins improve the likelihood of protein crystals forming. If examining pH then the possible effects of the buffer molecule on thermal stability should be taken into account along with the fact that pKa of each buffer molecule changes uniquely with temperature. Additionally, any time a charged species is examined the effects of the counterion should be accounted for. Thermal stability of proteins has traditionally been investigated using biochemical assays, circular dichroism, or differential scanning calorimetry. Biochemical assays require a catalytic activity of the protein in question as well as a specific assay. Circular dichroism and differential scanning calorimetry both consume large amounts of protein and are low-throughput methods. The thermofluor assay was the first high-throughput thermal shift assay and its utility and limitations has spurred the invention of a plethora of alternate methods. Each method has its strengths and weaknesses but they all struggle with intrinsically disordered proteins without any clearly defined tertiary structure as the essence of a thermal shift assay is measuring the temperature at which a protein goes from well-defined structure to disorder. ==Thermofluor== The technique was first described by Semisotnov et al. (1991) using 1,8-ANS and quartz cuvettes. Millenium Pharmaceuticals were the first to describe a high-throughput version using a plate reader and Wyeth Research published a variation of the method with SYPRO Orange instead of 1,8-ANS. SYPRO Orange has an excitation/emission wavelength profile compatible with qPCR machines which are almost ubiquitous in institutions that perform molecular biology research. The name Differential Scanning Fluorimetry (DSF) was introduced later but thermofluor is preferable as thermofluor is no longer trademarked and differential scanning fluorimetry is easily confused with differential scanning calorimetry. SYPRO Orange binds nonspecifically to hydrophobic surfaces, and water strongly quenches its fluorescence. When the protein unfolds, the exposed hydrophobic surfaces bind the dye, resulting in an increase in fluorescence by excluding water. Detergent micelles will also bind the dye and increase background noise dramatically - this effect is lessened by switching to the dye ANS but that requires UV excitation. The stability curve and its midpoint value (melting temperature, Tm also known as the temperature of hydrophobic exposure, Th) are obtained by gradually increasing the temperature to unfold the protein and measuring the fluorescence at each point. Curves are measured for protein only and protein + ligand, and ΔTm is calculated. The method might not work very well for protein-protein interactions if one of the interaction partners contains large hydrophobic patches as it is highly non-trivial to dissect prevention of aggregation, stabilisation of a native folds and steric hindrance of dye access to hydrophobic sites. In addition, partly aggregated protein can also limit the relative fluorescence increase upon heating; in extreme cases there will be no fluorescence increase at all because all protein is already in aggregates before heating. Knowing this effect can be very useful as a high relative fluorescence increase suggests a significant fraction of folded protein in the starting material. This assay allows high-throughput screening of ligands to the target protein and it is widely used in the early stages of drug discovery in the pharmaceutical industry, structural genomics efforts, and high-throughput protein engineering. 抄文引用元・出典: フリー百科事典『 ウィキペディア（Wikipedia）』 ■ウィキペディアで「Thermal shift assay」の詳細全文を読む スポンサード リンク
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