Evans T C, Nelsestuen G L
Department of Biochemistry, University of Minnesota, St. Paul, 55108, USA.
Biochemistry. 1996 Jun 25;35(25):8210-5. doi: 10.1021/bi9606354.
Upon addition of calcium to the metal-free protein, bovine prothrombin displays a conformational change with behavior of a classic trans- to cis-proline isomerization. The change is accompanied by a decrease of the intrinsic protein fluorescence and is essential to creating the membrane-binding conformation of prothrombin. This study showed that an identical conformational change was displayed by a peptide corresponding to residues 1-45 of prothrombin. This peptide contains a single tryptophan that underwent extensive quenching upon calcium addition. The kinetics were slow (t1/2 = 2.7 min at 24 degrees C) and displayed an activation energy of 24 kcal/mol. These properties overlapped precisely with the behavior of bovine prothrombin fragment 1 (residues 1-156). Consistent with studies on prothrombin and other vitamin K-dependent proteins that have been modified or truncated, the 1-45 peptide required about 10-fold higher calcium to elicit these behaviors than did fragment 1. The conformational change was necessary for membrane binding by the 1-45 peptide. The only proline in this sequence is at position 22. This proline is of the trans configuration in a crystallized form of calcium-bovine prothrombin fragment 1 [Soriano-Garcia, M., et al. (1992) Biochemistry 31, 2554]. Unless the protein conformational change is based on another behavior, this study showed that biochemical properties of the protein are inconsistent with structure solutions. Further studies are needed to reconcile structure/function in membrane association. Proline 22 in bovine prothrombin may constitute a useful biochemical marker for the membrane-binding conformation of a vitamin K-dependent protein.
在向无金属蛋白中添加钙后，牛凝血酶原会发生构象变化，呈现出典型的反式-顺式脯氨酸异构化行为。这种变化伴随着蛋白质固有荧光的降低，并且对于形成凝血酶原的膜结合构象至关重要。本研究表明，与凝血酶原1-45位残基对应的肽段也表现出相同的构象变化。该肽段含有一个单一的色氨酸，添加钙后会发生广泛的淬灭。动力学过程缓慢（24℃下t1/2 = 2.7分钟），活化能为24千卡/摩尔。这些特性与牛凝血酶原片段1（1-156位残基）的行为精确重叠。与对已修饰或截短的凝血酶原及其他维生素K依赖蛋白的研究一致，1-45肽段引发这些行为所需的钙比片段1高约10倍。构象变化对于1-45肽段的膜结合是必需的。该序列中唯一的脯氨酸位于22位。在钙-牛凝血酶原片段1的结晶形式中，这个脯氨酸是反式构型[Soriano-Garcia, M., et al. (1992) Biochemistry 31, 2554]。除非蛋白质的构象变化基于另一种行为，否则本研究表明蛋白质的生化特性与结构解析结果不一致。需要进一步研究来协调膜结合中的结构/功能关系。牛凝血酶原中的脯氨酸22可能构成一种有用的生化标记，用于指示维生素K依赖蛋白的膜结合构象。
