The Missing Electrostatic Interactions Between DNA Substrate and Sulfolobus solfataricus DNA Photolyase: What is the Role of Charged Amino Acids in Thermophilic DNA Binding Proteins?

Abstract

DNA photolyase can be used to study how a protein with its required cofactor has adapted over a large temperature range. The enzymatic activity and thermodynamics of substrate binding for protein from Sulfolobus solfataricus were directly compared to protein from Escherichia coli. Turnover numbers and catalytic activity were virtually identical, but organic cosolvents may be necessary to maintain activity of the thermophilic protein at higher temperatures. UV-damaged DNA binding to the thermophilic protein is less favorable by ∼2 kJ/mol. The enthalpy of binding is ∼10 kJ/mol less exothermic for the thermophile, but the amount and type of surface area buried upon DNA binding appears to be somewhat similar. The most important finding was observed when ionic strength studies were used to separate binding interactions into electrostatic and nonelectrostatic contributions; DNA binding to the thermophilic protein appears to lack the electrostatic contributions observed with the mesophilic protein.

Figure 1.

Absorption spectra of the FADH⁻ and FADH^· states of PL. Spectra were normalized for FADH^· absorbance. Solutions at 10 °C in 20 mM phosphate buffer pH 7.0 with 88 mM K₂SO₄.

Figure 2.

Typical ITC data and binding curve. Cell contained 240 μL of 36 μM FADH^· SsPL at 25 °C in 1000 mM ionic strength buffer, pH 7.0 and 280 rpm stirring. Syringe contained 350 μM UV-p(dT)₁₀ in same buffer. Substrate was added in 2.37 μL aliquots with 90 s per addition.

Figure 3.

Comparison of the Gibbs energy of binding from ITC. The values for EcPL as published earlier. Data obtained as described in the text. The FADH⁻ state is “red” and the FADH^· state is “SQ”. Errors for all points are reported in Table 1.

Figure 4.

Comparison of the enthalpy of binding. The values for EcPL as published earlier.15 Data obtained as described in the text. Dotted lines are the least-squares analysis of the data. Slopes (in kJ K⁻¹ mol⁻¹) for each: The FADH⁻ state is “red” and the FADH^· state is “SQ”. SsPL red = −820 (±100), SsPL SQ= −960 (±100), EcPL red = −1170 (±20), EcPL SQ = 1120 (±80). Individual errors are reported in Table 1.

Figure 5.

Comparison of ionic strength dependence of binding of ssDNA. ITC experiments were run in 50 mM Hepes, pH 7.0 with appropriate concentration of KCl. The FADH⁻ state is “red” and the FADH^· state is “SQ”. The dashed lines reflect the least-squares analysis of the data. The EcPL data as published previously. Uncertainty in data reported in Table 2.

Figure 6.

Interaction between CPD containing DNA and AnPL (1TEZ, ref 25).