Specific DNA recognition by the type II restriction endonuclease MunI: the effect of pH.
To investigate the effect of pH on sequence-specific binding, a thermodynamic characterization of the interaction of the protein MunI with a specific, and a nonspecific, oligonucleotide was performed. MunI is a type II restriction endonuclease which is able to bind specifically, but loses its enzymatic activity in the absence of magnesium ions. Comparison of the specific and nonspecific interactions at 10 and 25 degrees C shows that the latter is accompanied by a small change in enthalpy, and a negligible change in constant pressure heat capacity. On going through the pH range 5.75-9.0 at 25 degrees C, the affinity of specific complex formation is reduced by 20-fold. The interaction is accompanied by the protonation of groups assumed to be on the protein. Based on the simplest model that will fit the data, two distinct protonation events are observed. At low pH, two groups per protein molecule undergo protonation with a pK(a) of 6.0 and 6.9 in the free and bound forms, respectively. At high pH, a further independent protonation occurs involving two groups with pK(a) values of 8.9 and approximately 10.7 in the free and bound forms, respectively. The change in heat capacity ranges from -2.7 to -1.7 kJ mol(-1) K(-1) in going from pH 6.5 to 8.5. This range of variation of change in heat capacity can be accounted for by the effects of protonation of the interacting molecules. The change in heat capacity, calculated from surface area burial using a previously established relationship (1.15 kJ mol(-1) K(-1)), does not correlate well with the experimentally determined values.
Haq
I
I
Department of Biochemistry and Molecular Biology, University College London, Darwin Building, Gower Street, London, WC1E 6BT, U.K.
O'Brien
R
R
Lagunavicius
A
A
Siksnys
V
V
Ladbury
J E
JE
D002151:Calorimetry / Q000379:methods; D004247:DNA / Q000378:metabolism*; D015252:Deoxyribonucleases, Type II Site-Specific / Q000378:metabolism*; D004795:Enzyme Stability; D006863:Hydrogen-Ion Concentration; D009838:Oligodeoxyribonucleotides / Q000378:metabolism*; D011485:Protein Binding; D011522:Protons; D013696:Temperature; D013816:Thermodynamics
D009838:Oligodeoxyribonucleotides; D011522:Protons; D004247:DNA; C059776:endodeoxyribonuclease MfeI; D015252:Deoxyribonucleases, Type II Site-Specific