Zhu Mei M, Rempel Don L, Zhao Jiang, Giblin Daryl E, Gross Michael L
Department of Chemistry, Washington University, St. Louis, Missouri 63130, USA.
Biochemistry. 2003 Dec 30;42(51):15388-97. doi: 10.1021/bi035188o.
We applied a new method, "protein-ligand interaction using mass spectrometry, titration, and H/D exchange" (PLIMSTEX) [Zhu, M. M. (2003) J. Am. Chem. Soc. 125, 5252-5253], to determine the conformational changes, binding stoichiometry, and binding constants for Ca(2+) interactions with calmodulin (CaM) under varying conditions of electrolyte identity and ionic strength. The outcome shows that CaM becomes less solvent-accessible and more compact upon Ca(2+)-binding, as revealed by the PLIMSTEX curve. The formation of CaM-4Ca species is the biggest contributor to the shape of the titration curve, indicating that the formation of this species accounts for the largest conformational change in the stepwise Ca(2+) binding. The Ca(2+)-binding constants, when comparisons permit, agree with those in the literature within a factor of 3. The binding is influenced by ionic strength and the presence of other cations, although many of these cations do not cause conformational change in apo-CaM. Furthermore, Ca(2+)-saturated CaM exhibits larger protection and higher Ca(2+) affinity in media of low rather than high ionic strength. Both Ca(2+) and Mg(2+) bind to CaM with different affinities, causing different conformational changes. K(+), if it does bind, causes no detectable conformational change, and interactions of Ca(2+) with CaM in the presence of Li(+), Na(+), and K(+) occur with similar affinities and associated changes in solvent accessibility. These metal ion effects point to nonspecific rather than competitive binding of alkali-metal ions. The rates of deuterium uptake by the various CaM-xCa species follow a three-group (fast, intermediate, slow), pseudo-first-order kinetics model. Calcium binding causes the number of amide hydrogens to shift from the fast to the slow group. The results taken together not only provide new insight into CaM but also indicate that both PLIMSTEX and kinetic modeling of H/D exchange data may become general methods for probing protein conformations and quantifying protein-ligand interactions.
我们应用了一种新方法，即“利用质谱、滴定和氢/氘交换进行蛋白质-配体相互作用”（PLIMSTEX）[朱，M.M.（2003年）《美国化学会志》125，5252 - 5253]，来确定在电解质种类和离子强度不同的条件下，钙离子（Ca(2+)）与钙调蛋白（CaM）相互作用时的构象变化、结合化学计量比以及结合常数。结果表明，如PLIMSTEX曲线所示，Ca(2+)结合后CaM的溶剂可及性降低且结构更紧凑。CaM - 4Ca物种的形成对滴定曲线形状的贡献最大，表明该物种的形成在Ca(2+)逐步结合过程中导致了最大的构象变化。在可比较的情况下，Ca(2+)结合常数与文献中的值相差不超过3倍。结合受离子强度和其他阳离子的存在影响，尽管许多这些阳离子不会引起脱辅基CaM的构象变化。此外，Ca(2+)饱和的CaM在低离子强度而非高离子强度的介质中表现出更大的保护作用和更高的Ca(2+)亲和力。Ca(2+)和Mg(2+)以不同亲和力与CaM结合，导致不同的构象变化。钾离子（K(+)）如果确实结合，不会引起可检测到的构象变化，并且在锂离子（Li(+)）、钠离子（Na(+)）和钾离子存在的情况下，Ca(2+)与CaM的相互作用具有相似的亲和力以及相关的溶剂可及性变化。这些金属离子效应表明碱金属离子的结合是非特异性的而非竞争性的。各种CaM - xCa物种的氘摄取速率遵循三组（快、中、慢）伪一级动力学模型。钙离子结合导致酰胺氢的数量从快速组转移到慢速组。综合这些结果不仅为CaM提供了新的见解，还表明PLIMSTEX以及氢/氘交换数据的动力学建模可能成为探测蛋白质构象和量化蛋白质-配体相互作用的通用方法。
