System of measuring the instability of a protein under varying conditions
A thermal shift assay (TSA) measures changes in the thermal denaturation temperature and hence stability of a protein under varying conditions such as variations in drug concentration, buffer pH or ionic strength, redox potential, or sequence mutation. The most common method for measuring protein thermal shifts is differential scanning fluorimetry (DSF) or thermofluor, which utilizes specialized fluorogenic dyes.[1]
The binding of low molecular weight ligands can increase the thermal stability of a protein, as described by Daniel Koshland (1958)[2] and Kaj Ulrik Linderstrøm-Lang and Schellman (1959).[3] Almost half of enzymes require a metal ion co-factor.[4] Thermostable proteins are often more useful than their non-thermostable counterparts, e.g., DNA polymerase in the polymerase chain reaction,[5] so protein engineering often includes adding
mutations to increase thermal stability. Protein crystallization is more successful for proteins with a higher melting point[6] and adding buffer components that stabilize proteins improve the likelihood of protein crystals forming.[7]
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.[8] 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.
^Koshland DE (February 1958). "Application of a Theory of Enzyme Specificity to Protein Synthesis". Proceedings of the National Academy of Sciences of the United States of America. 44 (2): 98–104. Bibcode:1958PNAS...44...98K. doi:10.1073/pnas.44.2.98. PMC 335371. PMID 16590179.
^Linderstrøm-Lang K, Schellman JA (1959). "Protein structure and enzyme activity". The Enzymes. 1 (2): 443–510.
^Waldron KJ, Rutherford JC, Ford D, Robinson NJ (August 2009). "Metalloproteins and metal sensing". Nature. 460 (7257): 823–30. Bibcode:2009Natur.460..823W. doi:10.1038/nature08300. PMID 19675642. S2CID 205217922.
^Saiki RK, Gelfand DH, Stoffel S, Scharf SJ, Higuchi R, Horn GT, et al. (January 1988). "Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase". Science. 239 (4839): 487–91. Bibcode:1988Sci...239..487S. doi:10.1126/science.239.4839.487. PMID 2448875.
^Dupeux F, Röwer M, Seroul G, Blot D, Márquez JA (November 2011). "A thermal stability assay can help to estimate the crystallization likelihood of biological samples". Acta Crystallographica. Section D, Biological Crystallography. 67 (Pt 11): 915–9. doi:10.1107/s0907444911036225. PMID 22101817.
^Ericsson UB, Hallberg BM, Detitta GT, Dekker N, Nordlund P (October 2006). "Thermofluor-based high-throughput stability optimization of proteins for structural studies". Analytical Biochemistry. 357 (2): 289–98. doi:10.1016/j.ab.2006.07.027. PMID 16962548.
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