Sui G C, Wiman B
Department of Clinical Chemistry and Blood Coagulation, Karolinska Hospital, Karolinska Institute, Stockholm, Sweden.
Thromb Haemost. 2000 Jun;83(6):896-901.
We have investigated the B beta-sheet in PAI-1 regarding its role for the stability of the molecule. The residues from His(219) to Tyr(241) (except for Gly(230) and Pro(240)), covering the s2B and s3B strands, and in addition His(185) and His(190)) were substituted by amino acids with opposite properties. The 23 generated single-site changed mutants and also wild type PAI-1 (wtPAI-1) were expressed in E. coli. Subsequently they were purified by heparin-Sepharose and anhydrotrypsin agarose affinity chromatographies. The stability of the purified PAI-1 variants was analyzed at 37 degrees C and at different pHs (5.5, 6.5 or 7.5). At pH 7.5 and 37 degrees C, single substitutions of the residues in the central portions of both strands 2 and 3 in the B beta-sheet (Ile(223) to Leu(226) on s2B and Met(235) to Ile(237) on s3B), caused a significant decrease in stability, yielding half-lives of about 10-25% as compared to wtPAI-1. On the other hand, mutations at both sides of the central portion of the B beta-sheet (Tyr(221), Asp(222), Tyr(228) and Thr(232)) frequently resulted in an increased PAI-1 stability (up to 7-fold). While wtPAI-1 exhibited prolonged half-lives at pH 6.5 and 5.5, the PAI-1 variant Y228S was more stable at neutral pH (half-life of 9.6 h at pH 7.5) as compared to its half-life at pH 5.5 (1.1 h). One of the 4 modified histidine residues (His(229)) resulted in a variant with a clearly affected stability as a function of pH, suggesting that it may, at least in part, be of importance for the pH dependence of the PAI-1 stability. Thus, our data demonstrate that the B beta-sheet is of great importance for the stability of the molecule. Modifications in this part causes decreased or increased stability in a certain pattern, suggesting effects on the insertion rate of the reactive center loop into the A beta-sheet of the molecule.
我们研究了纤溶酶原激活物抑制剂-1（PAI-1）中的Bβ折叠对分子稳定性的作用。覆盖s2B和s3B链的从组氨酸（His）(219)到酪氨酸（Tyr）(241)的残基（除甘氨酸（Gly）(230)和脯氨酸（Pro）(240)外），以及另外的组氨酸（His）(185)和组氨酸（His）(190)）被具有相反性质的氨基酸取代。所产生的23个单点突变体以及野生型PAI-1（wtPAI-1）在大肠杆菌中表达。随后通过肝素-琼脂糖凝胶和脱水胰蛋白酶琼脂糖亲和层析进行纯化。在37℃和不同pH值（5.5、6.5或7.5）下分析纯化的PAI-1变体的稳定性。在pH 7.5和37℃时，Bβ折叠中链2和链3中央部分的残基单点取代（s2B上的异亮氨酸（Ile）(223)至亮氨酸（Leu）(226)以及s3B上的甲硫氨酸（Met）(235)至异亮氨酸（Ile）(237)）导致稳定性显著降低，与wtPAI-1相比，半衰期约为10 - 25%。另一方面，Bβ折叠中央部分两侧的突变（酪氨酸（Tyr）(221)、天冬氨酸（Asp）(222)、酪氨酸（Tyr）(228)和苏氨酸（Thr）(232)）经常导致PAI-1稳定性增加（高达7倍）。虽然wtPAI-1在pH 6.5和5.5时半衰期延长，但PAI-1变体Y228S在中性pH（pH 7.5时半衰期为9.6小时）下比其在pH 5.5时（1.1小时）更稳定。4个修饰的组氨酸残基之一（组氨酸（His）(229)）产生了一个稳定性明显受pH影响的变体，表明它可能至少部分地对PAI-1稳定性的pH依赖性很重要。因此，我们的数据表明Bβ折叠对分子稳定性非常重要。这部分的修饰会以某种模式导致稳定性降低或增加，提示对反应中心环插入分子Aβ折叠的速率有影响。
