Thermal stability landscape for Klenow DNA polymerase as a function of pH and salt concentration.
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 degrees C, from 40 to 62 degrees 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.
Richard
Allison J
AJ
Department of Biological Sciences, Louisiana State University, Baton Rouge, LA 70803, USA.
Liu
Chin-Chi
CC
Klinger
Alexandra L
AL
Todd
Matthew J
MJ
Mezzasalma
Tara M
TM
LiCata
Vince J
VJ
D000817:Anilino Naphthalenesulfonates / Q000737:chemistry; D002413:Cations, Divalent / Q000737:chemistry; D002414:Cations, Monovalent / Q000737:chemistry; D004256:DNA Polymerase I / Q000737:chemistry*; D004795:Enzyme Stability; D005453:Fluorescence; D006863:Hydrogen-Ion Concentration; D011487:Protein Conformation; D012492:Salts / Q000737:chemistry; D013050:Spectrometry, Fluorescence; D044366:Transition Temperature
D000817:Anilino Naphthalenesulfonates; D002413:Cations, Divalent; D002414:Cations, Monovalent; D012492:Salts; C035763:5,5'-bis(8-(phenylamino)-1-naphthalenesulfonate); D004256:DNA Polymerase I