The thermal denaturation of Klenow DNA polymerase has been characterized over a wide variety of solution conditions to obtain a relative stability landscape for the protein. Measurements were conducted utilizing a miniaturized fluorescence assay that measures Tm based on the increase in the fluorescence of 1,8-anilinonaphthalene sulfonate (ANS) when the protein denatures. The melting temperature (Tm) for Klenow increases as the salt concentration is increased and as the pH is decreased. Klenow's Tm spans a range of over 20 °C, from 40 to 62 °C, depending upon the solution conditions. The landscape reconciles and extends previously measured Tm values for Klenow. Salt effects on the stability of Klenow show strong cation dependence overlaid onto a more typical Hofmeister anion type dependence. Cationic stabilization of proteins has been far less frequently documented than anionic stabilization. The monovalent cations tested stabilize Klenow with the following hierarchy: NH4+ > Na+ > Li+ > K+. Of the divalent cations tested: Mg+2 and Mn+2 significantly stabilize the protein, while Ni+2 dramatically destabilizes the protein. Stability measurements performed in combined Mg+2 plus Na+ salts suggest that the stabilizing effects of these monovalent and divalent cations are synergistic. The cationic stabilization of Klenow can be well explained by a model postulating dampening of repulsion within surface anionic patches on the protein. © 2006 Elsevier B.V. All rights reserved.
Publication Source (Journal or Book title)
Biochimica et Biophysica Acta - Proteins and Proteomics
Richard, A., Liu, C., Klinger, A., Todd, M., Mezzasalma, T., & LiCata, V. (2006). Thermal stability landscape for Klenow DNA polymerase as a function of pH and salt concentration. Biochimica et Biophysica Acta - Proteins and Proteomics, 1764 (10), 1546-1552. https://doi.org/10.1016/j.bbapap.2006.08.011