STUDY OF STRONG TO ULTRATIGHT PROTEIN INTERACTIONS USING DIFFERENTIAL SCANNING CALORIMETRY
STUDY OF STRONG TO ULTRATIGHT PROTEIN INTERACTIONS USING DIFFERENTIAL SCANNING CALORIMETRY
复制标题
DOI：
10.1021/bi00481a024
复制
发表时间：
1990-07-24
期刊：
影响因子：
2.9
通讯作者：
LIN, LN
中科院分区：
文献类型：
--
作者：
BRANDTS, JF;LIN, LN
差示扫描量热法 (DSC) 的数据可用于估计无法通过更传统的平衡技术方便测量的非常大的结合常数。热力学模型已被制定来描述涉及一个热转变（蛋白质-配体）或两个热转变（蛋白质-蛋白质）以及 1:1 或更高结合化学计量的相互作用系统。描述了通过两种不同方法获得结合常数和结合热的方法：数据的计算或模拟拟合。通过这两种方法提供并分析了 2''CMP 与 RNase 结合的大量 DSC 数据。结果发现，当结合位点完全饱和时，计算方法是一致的，但当位点饱和不完全且配体分子的转变与未配体分子的转变重叠时，计算方法会产生很大的误差。这主要是由于无法在弱结合条件下确定 TM（即折叠和未折叠蛋白质浓度相等的温度）。模拟结果表明，当外推到相同温度时，DSC 方法的结合常数和结合热与平衡方法获得的相应估计值在定量上一致。从DSC数据还发现，结合常数随着配体浓度的增加而降低，这可能是由与2''CMP二聚化相关的非理想效应引起的。模拟表明 DSC 方法能够估计高达 1040 M-1 或更高的超紧密相互作用的结合常数，而大多数平衡方法在远低于 1010 M-1 时会失败。来自许多相互作用系统的文献中的 DSC 数据（胰蛋白酶-大豆胰蛋白酶抑制剂、胰蛋白酶-卵类粘蛋白、胰蛋白酶-胰腺胰蛋白酶抑制剂、糜蛋白酶-枯草杆菌蛋白酶抑制剂、枯草杆菌蛋白酶 BPN-枯草杆菌蛋白酶抑制剂、RNase S 蛋白-RNase S 肽、亲和素-生物素、卵转铁蛋白-Fe3+、 通过模拟拟合或计算来分析超氧化物歧化酶-Zn2+、碱性磷酸酶-Zn2+以及天冬氨酸转氨甲酰酶调节和催化亚基的组装。表观单位点结合常数范围为约。 1-5 至 1020 M-1，而天冬氨酸转氨甲酰酶组装的相互作用常数以摩尔浓度单位估计为 1037。对于大多数这些系统，DSC 相互作用常数与其他文献估计相比有利，对于某些系统，由于未知原因没有达到预期，而对于其他系统，这代表了第一个估计值。模拟表明，对于在单个协作单元内具有相同配体的两个结合位点的蛋白质，当接近未配体蛋白质的转变温度时，配体重排将在 DSC 扫描期间自发发生。趋势是形成比在较低温度下存在的更多的未配体和双配体蛋白质，这导致不存在单配体形式的转变。尽管这种趋势始终存在，但在配体重排动力学相对于实验扫描速率较慢的系统中可能不会表达，在这种情况下，配体分布将冻结在低温构型中，并且可能会看到单配体形式的转变。对于所检查的四个此类系统，只有一个（卵转铁蛋白-Fe3+）在扫描过程中似乎保持低温配置。
Data from differential scanning calorimetry (DSC) may be used to estimate very large binding constants that cannot be conveniently measured by more conventional equilibrium techniques. Thermodynamic models have been formulated to describe interacting systems that involve either one thermal transition (protein-ligand) or two thermal transitions (protein-protein) and either 1:1 or higher binding stoichiometry. Methods are described for obtaining binding constants and heats of binding by two different methods: calculation or simulation fitting of data. Extensive DSC data on 2''CMP binding to RNase are presented and analyzed by the two methods. It is found that the methods agree when binding sites are completely saturated, but substantial errors arise in the calculation method when site saturation is incomplete and the transition of liganded molecules overlaps that of unliganded molecules. This arises primarily from an inability to determine TM (i.e., the temperature where concentrations of folded and unfolded protein are equal) under weak-binding conditions. Results from simulation show that the binding constants and heats of binding from the DSC method agree quantitatively with corresponding estimates obtained from equilibrium methods when extrapolated to the same temperature. It was also found from the DSC data that the binding constant decreases with increasing concentration of ligand, which might arise from nonideality effects associated with dimerization of 2''CMP. Simulations show that the DSC method is capable of estimating binding constants for ultratight interactions up to perhaps 1040 M-1 or higher, while most equilibrium methods fail well below 1010 M-1. DSC data from literature on a number of interacting systems (trypsin-soybean trypsin inhibitor, trypsin-ovomucoid, trypsin-pancreatic trypsin inhibitor, chymotrypsin-subtilisin inhibitor, subtilisin BPN-subtilisin inhibitor, RNase S protein-RNase S peptide, avidin-biotin, ovotransferrin-Fe3+, superoxide dismutase-Zn2+, alkaline phosphatase-Zn2+, and assembly of regulatory and catalytic subunits of aspartate transcarbamoylase) were analyzed by simulation fitting or by calculation. Apparent single-site binding constant ranged from ca. 1-5 to 1020 M-1, while the interaction constant for assembly of aspartate transcarbamoylase was estimated as 1037 in molarity units. For most of these systems, the DSC interaction constants compared favorably with other literature estimates, for some it did not for reasons unknown, while for still others this represented the first estimated. Simulations show that for proteins having two binding sites for the same ligand within a single cooperative unit, ligand rearrangement will occur spontaneously during a DSC scan as the transition temperature of the unliganded protein is approached. The tendency is to form more of the unliganded and doubly liganded protein than is present at lower temperatures, which the leads to the absence of a transition for the singly liganded form. Although this tendency will always exist, it may not be expressed in systems where the kinetics for ligannd rearrangement are slow relative to the experimental scan rate, in which case the ligand distribution will be frozen in the low-temperature configuration and a transition for the singly liganded form(s) might be seen. For the four systems of this type that were examined, only one (ovotransferrin-Fe3+) appeared to remain in a low-temperature configuration during scanning.