Almeida Paulo F F, Sohma Hitoshi, Rasch Katie A, Wieser Catherine M, Hinderliter Anne
Department of Chemistry and Biochemistry, University of North Carolina-Wilmington, Wilmington North Carolina 28403,
Biochemistry. 2005 Aug 16;44(32):10905-13. doi: 10.1021/bi050474g.
Annexins are a family of proteins generally described as Ca(2+)-dependent for phospholipid binding. Yet, annexins have a wide variety of binding behaviors and conformational states, some of which are lipid-dependent and Ca(2+)-independent. We present a model that captures the cation and phospholipid binding behavior of the highly conserved core of the annexins. Experimental data for annexins A4 and A5, which have short N-termini, were globally modeled to gain an understanding of how the lipid-binding affinity of the conserved protein core is modulated. Analysis of the binding behavior was achieved through use of the lanthanide Tb(3+) as a Ca(2+) analogue. Binding isotherms were determined experimentally from the quenching of the intrinsic fluorescence of annexins A4 and A5 by Tb(3+) in the presence or absence of membranes. In the presence of lipid, the affinity of annexin for cation increases, and the binding isotherms change from hyperbolic to weakly sigmoidal. This behavior was modeled by isotherms derived from microscopic binding partition functions. The change from hyperbolic to sigmoidal binding occurs because of an allosteric transition from the annexin solution state to its membrane-associated state. Protein binding to lipid bilayers renders cation binding by annexins cooperative. The two annexin states denote two affinities of the protein for cation, one in the absence and another in the presence of membrane. In the framework of this model, we discuss membrane binding as well as the influence of the N-terminus in modifying the annexin cation-binding affinity by changing the probability of the protein to undergo the postulated two-state transition.
膜联蛋白是一类通常被描述为依赖Ca(2+)进行磷脂结合的蛋白质家族。然而，膜联蛋白具有多种结合行为和构象状态，其中一些是脂质依赖性且不依赖Ca(2+)的。我们提出了一个模型，该模型能够捕捉膜联蛋白高度保守核心的阳离子和磷脂结合行为。对具有短N端的膜联蛋白A4和A5的实验数据进行了全局建模，以了解保守蛋白核心的脂质结合亲和力是如何被调节的。通过使用镧系元素Tb(3+)作为Ca(2+)类似物来分析结合行为。结合等温线是在有或没有膜存在的情况下，通过Tb(3+)对膜联蛋白A4和A5内在荧光的淬灭实验测定的。在有脂质存在的情况下，膜联蛋白对阳离子的亲和力增加，结合等温线从双曲线型变为弱S型。这种行为通过从微观结合分配函数导出的等温线进行建模。结合从双曲线型变为S型是由于膜联蛋白从溶液状态到其膜相关状态的变构转变。蛋白质与脂质双层的结合使膜联蛋白的阳离子结合具有协同性。膜联蛋白的两种状态表示蛋白质对阳离子的两种亲和力，一种在没有膜时，另一种在有膜时。在这个模型的框架内，我们讨论了膜结合以及N端通过改变蛋白质经历假定的双态转变的概率来改变膜联蛋白阳离子结合亲和力的影响。
