Cimmperman Piotras, Baranauskiene Lina, Jachimoviciūte Simona, Jachno Jelena, Torresan Jolanta, Michailoviene Vilma, Matuliene Jurgita, Sereikaite Jolanta, Bumelis Vladas, Matulis Daumantas
Laboratory of Biothermodynamics and Drug Design, Institute of Biotechnology, LT-02241 Vilnius, Lithuania.
Biophys J. 2008 Oct;95(7):3222-31. doi: 10.1529/biophysj.108.134973. Epub 2008 Jul 3.
Equilibrium binding ligands usually increase protein thermal stability by an amount proportional to the concentration and affinity of the ligand. High-throughput screening for the discovery of drug-like compounds uses an assay based on thermal stabilization. The mathematical description of this stabilization is well developed, and the method is widely applicable to the characterization of ligand-protein binding equilibrium. However, numerous cases have been experimentally observed where equilibrium binding ligands destabilize proteins, i.e., diminish protein melting temperature by an amount proportional to the concentration and affinity of the ligand. Here, we present a thermodynamic model that describes ligand binding to the native and unfolded (denatured) protein states explaining the combined stabilization and destabilization effects. The model also explains nonsaturation and saturation effects on the protein melting temperature when the ligand concentration significantly exceeds the protein concentration. Several examples of the applicability of the model are presented, including specific sulfonamide binding to recombinant hCAII, peptide and ANS binding to the Polo-box domain of Plk1, and zinc ion binding to the recombinant porcine growth hormone. The same ligands may stabilize and destabilize different proteins, and the same proteins may be stabilized and destabilized by different ligands.
平衡结合配体通常会使蛋白质的热稳定性提高，提高的幅度与配体的浓度和亲和力成正比。用于发现类药物化合物的高通量筛选采用基于热稳定性的分析方法。这种稳定性的数学描述已经很完善，该方法广泛适用于配体 - 蛋白质结合平衡的表征。然而，实验中观察到许多情况，即平衡结合配体使蛋白质不稳定，也就是说，使蛋白质的解链温度降低，降低的幅度与配体的浓度和亲和力成正比。在此，我们提出一个热力学模型，该模型描述了配体与天然和未折叠（变性）蛋白质状态的结合，解释了稳定和不稳定的综合效应。该模型还解释了当配体浓度显著超过蛋白质浓度时，对蛋白质解链温度的非饱和和饱和效应。文中给出了该模型适用性的几个例子，包括特定磺酰胺与重组人碳酸酐酶II的结合、肽和ANS与Plk1的Polo盒结构域的结合，以及锌离子与重组猪生长激素的结合。相同的配体可能使不同的蛋白质稳定或不稳定，相同的蛋白质也可能被不同的配体稳定或不稳定。
