ON THE BIOSYNTHESIS OF BOVINE PANCREATIC TRYPSIN-INHIBITOR (BPTI) - STRUCTURE, PROCESSING, FOLDING AND DISULFIDE BOND FORMATION OF THE PRECURSOR IN-VITRO AND IN MICROSOMES
ON THE BIOSYNTHESIS OF BOVINE PANCREATIC TRYPSIN-INHIBITOR (BPTI) - STRUCTURE, PROCESSING, FOLDING AND DISULFIDE BOND FORMATION OF THE PRECURSOR IN-VITRO AND IN MICROSOMES
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DOI：
10.1006/jmbi.1993.1470
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发表时间：
1993-08-20
影响因子：
5.6
通讯作者：
SHEIKH, A
中科院分区：
文献类型：
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作者：
CREIGHTON, TE;BAGLEY, CJ;SHEIKH, A
牛胰蛋白酶抑制剂 (BPTI) 的天然基因通过体外转录/翻译系统表达为 100 个残基的前 proBPTI，并带有用于易位到内质网的信号肽。在微粒体存在下的表达定义了信号肽共翻译切割的位点。微粒体中所得的 proBPTI 由成熟 BPTI 的 58 个残基以及 N 末端的另外 13 个残基组成，其中包括第 10 位的半胱氨酸残基和 C 末端的 7 个残基。在还原条件下合成时，ProBPTI 在微粒体中保持未折叠的还原形式，但当添加二硫键形式的谷胱甘肽时，ProBPTI 会快速折叠并形成二硫键。即使当残基Cys10被Ser取代时，大约一分钟内即可发生完全折叠。通过圆二色性和二维NMR确定proBPTI的结构，发现其是在两个末端具有柔性延伸的成熟BPTI的结构。它对 α-胰凝乳蛋白酶活性的抑制与成熟蛋白的抑制没有区别。在折叠和二硫键形成耦合的条件下，前体的延伸似乎在体外重折叠中仅发挥非常小的作用。在被认为反映体内条件的 pH 和氧化还原条件下，完全折叠和二硫键形成需要几个小时。在体外折叠实验中添加蛋白质二硫化物异构酶导致成熟 BPTI 和 proBPTI 完全折叠状态形成速率的显着且相似的增加；折叠到天然状态的半时间减少到大约两分钟，这与微粒体中发生的时间相当。由于 N 和 C 末端延伸对蛋白质结构、抑制剂活性和重折叠没有实质性影响，因此它们的功能作用有待发现。
The natural gene for bovine pancreatic trypsin inhibitor (BPTI) was expressed byin vitrotranscription/translation systems as the 100-residue pre-proBPTI, with a signal peptide for translocation into the endoplasmic reticulum. Expression in the presence of microsomes defined the site of co-translational cleavage of the signal peptide. The resulting proBPTI in the microsomes consists of the 58 residues of mature BPTI, plus an additional 13 residues at the N terminus, including a cysteine residue at position - 10, and seven residues at the C terminus. ProBPTI remained in the unfolded, reduced form within microsomes when synthesized under reducing conditions, but folded and formed disulphide bonds rapidly when the disulphide form of glutathione was added. Complete folding could occur within about one minute, even when residue Cys10 was replaced by Ser.The structure of proBPTI was determined by circular dichroism and two-dimensional NMR and found to be that of mature BPTI with flexible extensions on both termini. Its inhibition of the activity of α-chymotrypsin was indistinguishable from that of the mature protein. The extensions of the precursor appeared to play only very minor roles in refoldingin vitrounder conditions where folding and disulphide bond formation are coupled. Under pH and redox conditions thought to reflect thosein vivo, complete folding and disulphide bond formation required several hours. Addition of protein disulphide isomerase toin vitrofolding experiments caused substantial and similar increases in the rate of formation of the fully folded state for both mature BPTI and proBPTI; the half time for folding to the native state was reduced to approximately two minutes, which is comparable to that occurring in microsomes. The absence of substantial effects of the N and C-terminal extensions on the protein structure, inhibitor activity and refolding leaves their functional roles to be discovered.