Moll Daniela, Schweinsberg Sonja, Hammann Christian, Herberg Friedrich W
Department of Biochemistry, University of Kassel, Heinrich-Plett-Str. 40, D-34132 Kassel, Germany.
Biol Chem. 2007 Feb;388(2):163-72. doi: 10.1515/BC.2007.018.
We have investigated the thermodynamic parameters and binding of a regulatory subunit of cAMP-dependent protein kinase (PKA) to its natural low-molecular-weight ligand, cAMP, and analogues thereof. For analysis of this model system, we compared side-by-side isothermal titration calorimetry (ITC) with surface plasmon resonance (SPR). Both ITC and SPR analyses revealed that binding of the protein to cAMP or its analogues was enthalpically driven and characterised by similar free energy values (DeltaG=-9.4 to -10.7 kcal mol-1) for all interactions. Despite the similar affinities, binding of the cyclic nucleotides used here was characterised by significant differences in the contribution of entropy (-TDeltaS) and enthalpy (DeltaH) to DeltaG. The comparison of ITC and SPR data for one cAMP analogue further revealed deviations caused by the method. These equilibrium parameters could be complemented by thermodynamic data of the transition state (DeltaHnot equal, DeltaGnot equal, DeltaSnot equal) for both association and dissociation measured by SPR. This direct comparison of ITC and SPR highlights method-specific advantages and drawbacks for thermodynamic analyses of protein/ligand interactions.
我们研究了环磷酸腺苷（cAMP）依赖性蛋白激酶（PKA）的一个调节亚基与其天然低分子量配体cAMP及其类似物的热力学参数和结合情况。为了分析这个模型系统，我们将等温滴定量热法（ITC）与表面等离子体共振（SPR）进行了并列比较。ITC和SPR分析均表明，该蛋白与cAMP或其类似物的结合是由焓驱动的，并且所有相互作用的自由能值相似（ΔG = -9.4至-10.7 kcal mol-1）。尽管亲和力相似，但此处使用的环核苷酸的结合在熵（-TΔS）和焓（ΔH）对ΔG的贡献方面存在显著差异。对一种cAMP类似物的ITC和SPR数据的比较进一步揭示了方法导致的偏差。这些平衡参数可以通过SPR测量的缔合和解离的过渡态热力学数据（ΔH≠、ΔG≠、ΔS≠）进行补充。ITC和SPR的这种直接比较突出了蛋白质/配体相互作用热力学分析中方法特定的优点和缺点。
