Ellison E H, Castellino F J
Department of Chemistry and Biochemistry, University of Notre Dame, Indiana 46556, USA.
Biochemistry. 1998 Jun 2;37(22):7997-8003. doi: 10.1021/bi973118+.
Spread phospholipid monolayers are particularly useful as model membranes in that changes in surface pressure (Deltapi) can be monitored in response to protein adsorption to the monolayer, thus providing a unique manner of assessing protein-membrane contact. In the present study, spread monolayers below their collapse pressures have been utilized to evaluate Ca2+-specific adsorption of several vitamin K-dependent coagulation proteins to monolayers that contain negatively charged phospholipid. From combined measurements of Deltapi and Gamma (the surface excess protein concentration), values of dGamma/dpi have been evaluated for different proteins with varying lipid composition of the monolayers. Using mixed, liquid-expanded monolayers at equivalent initial surface pressures (pii) and which contain different amounts of phosphatidylserine, phosphatidylcholine, and phosphatidylethanolamine, the dGamma/dpi of bovine prothrombin was shown to decrease monotonically with increasing protein affinity for the monolayer. For example, KD values of 7, 20, and 60 nM produced dGamma/dpi values of 14, 17, and 21 nmol m-1 mN-1, respectively. However, the trend in dGamma/dpi appears to originate from characteristics of the monolayer and not from those of the protein, since a much different adsorbate (i.e., a positively charged pyrene derivative) exhibited a similar trend in dGamma/dpi with monolayer composition. On the other hand, dGamma/dpi values of bovine prothrombin, human factor IX, human protein S, bovine protein C, and human protein C, determined using liquid-expanded phosphatidylserine monolayers, were essentially equivalent. Therefore, the five vitamin K-dependent proteins that were examined were equivalent in terms of the manner in which the gamma-carboxyglutamic acid (Gla) domain of each protein perturbed the surface pressure. This study shows that Ca2+-specific membrane contact sites in the Gla domain of the five proteins tested are similar despite the naturally occurring differences in the normal Gla domain sequence of these proteins.
铺展磷脂单分子层作为模型膜特别有用，因为可以监测表面压力（Δπ）的变化以响应蛋白质吸附到单分子层上，从而提供一种评估蛋白质 - 膜接触的独特方式。在本研究中，低于其崩塌压力的铺展单分子层已被用于评估几种维生素K依赖性凝血蛋白对含有带负电荷磷脂的单分子层的Ca2 +特异性吸附。通过对Δπ和Γ（表面过量蛋白质浓度）的联合测量，已针对具有不同脂质组成的单分子层的不同蛋白质评估了dΓ/dπ值。使用在等效初始表面压力（pii）下且含有不同量磷脂酰丝氨酸、磷脂酰胆碱和磷脂酰乙醇胺的混合液体膨胀单分子层，牛凝血酶原的dΓ/dπ显示随着蛋白质对单分子层亲和力的增加而单调降低。例如，7、20和60 nM的KD值分别产生14、17和21 nmol m-1 mN-1的dΓ/dπ值。然而，dΓ/dπ的趋势似乎源自单分子层的特性而非蛋白质的特性，因为一种非常不同的吸附物（即带正电荷的芘衍生物）在dΓ/dπ与单分子层组成方面表现出类似的趋势。另一方面，使用液体膨胀的磷脂酰丝氨酸单分子层测定的牛凝血酶原、人因子IX、人蛋白S、牛蛋白C和人蛋白C的dΓ/dπ值基本相当。因此，所检测的五种维生素K依赖性蛋白质在每种蛋白质的γ-羧基谷氨酸（Gla）结构域扰动表面压力的方式方面是等效的。这项研究表明，尽管这些蛋白质的正常Gla结构域序列存在天然差异，但所测试的五种蛋白质的Gla结构域中的Ca2 +特异性膜接触位点是相似的。
