Hackeng T M, Dawson P E, Kent S B, Griffin J H
Department of Molecular and Experimental Medicine, Scripps Research Institute, La Jolla, CA 92037, USA.
Biopolymers. 1998 Aug;46(2):53-63. doi: 10.1002/(SICI)1097-0282(199808)46:2<53::AID-BIP1>3.0.CO;2-W.
Human plasma protein S is a nonenzymatic cofactor for activated protein C (APC) in the inactivation of coagulation factors Va and VIIIa, and helps to provide an essential negative feedback on blood coagulation. Previous indirect evidence suggested that the thrombin-sensitive region (TSR: residues 47-75, 1 disulfide) and the first epidermal growth factorlike region (EGF1: residues 76-116, 3 disulfides) of protein S may be functionally important for expression of its APC cofactor activity. To study the functional importance of these modules directly, access to the isolated TSR and EGF1 modules would be preferred. Recombinant expression of protein S intact TSR and correctly folded EGF1 has not been possible. Here we describe the synthesis of both TSR and EGF1 modules by stepwise solid phase peptide synthesis using the in situ neutralization/2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluron ium hexafluorophosphate activation procedure for tert-butoxycarbonyl chemistry. For the TSR, correct intramodular disulfide bonding was confirmed. To overcome folding difficulties with the EGF1, a two-step oxidation procedure was used in which the cysteines involved in the middle, crossing, disulfide bond (Cys85-Cys102) remained protected with acetamidomethyl (Acm) groups after hydrogen fluoride treatment of the peptide resin. Selective formation of the first two disulfide bonds (Cys80-Cys93 and Cys104-Cys113) was followed by release of the Acm groups and subsequent formation of the third disulfide bond (Cys85-Cys102). CD studies revealed 54% of beta-sheet/turn in the EGF1 that is characteristic for EGF modules. Deuterium exchange studies suggested a very tightly packed core in EGF1 that is not accessible to the bulk solvent, likely a result from the compact structure caused by its three disulfide bonds. The 30% beta-sheet structure observed in the TSR involved amide protons that could be readily exchanged by deuterons, likely reflecting a more flexible structure of the TSR loop in contrast to the rigid structure of EGF1. The establishment of synthetic access to the TSR and EGF1 of protein S provides a versatile tool to study interactions of these modules with the blood coagulation components of the anticoagulant plasma protein C pathway.
人血浆蛋白S是活化蛋白C（APC）使凝血因子Va和VIIIa失活过程中的非酶辅因子，有助于对血液凝固提供重要的负反馈。先前的间接证据表明，蛋白S的凝血酶敏感区（TSR：第47 - 75位氨基酸残基，1个二硫键）和首个表皮生长因子样区域（EGF1：第76 - 116位氨基酸残基，3个二硫键）对于其APC辅因子活性的表达可能在功能上很重要。为了直接研究这些模块的功能重要性，获取分离的TSR和EGF1模块会更可取。完整的蛋白S TSR和正确折叠的EGF1的重组表达一直无法实现。在此，我们描述了通过逐步固相肽合成法合成TSR和EGF1模块，使用原位中和/2 -（1H - 苯并三唑 - 1 - 基）- 1,1,3,3 - 四甲基脲六氟磷酸盐活化程序进行叔丁氧羰基化学合成。对于TSR，确认了模块内正确的二硫键连接。为了克服EGF1的折叠困难，采用了两步氧化程序，其中在肽树脂经氟化氢处理后，参与中间交叉二硫键（Cys85 - Cys102）的半胱氨酸仍被乙酰氨基甲基（Acm）基团保护。先选择性形成前两个二硫键（Cys80 - Cys93和Cys104 - Cys113），然后释放Acm基团并随后形成第三个二硫键（Cys85 - Cys102）。圆二色性（CD）研究显示EGF1中有54%的β - 折叠/转角结构，这是EGF模块的特征。氘交换研究表明EGF1中有一个非常紧密堆积的核心，大量溶剂无法进入，这可能是其由三个二硫键导致的紧密结构的结果。在TSR中观察到的30%的β - 折叠结构涉及酰胺质子，这些质子很容易被氘交换，这可能反映了与EGF1的刚性结构相比，TSR环的结构更灵活。建立对蛋白S的TSR和EGF1的合成途径，为研究这些模块与抗凝血浆蛋白C途径的血液凝固成分之间的相互作用提供了一个通用工具。
