Reduced BPTI is collapsed. A pulsed field gradient NMR study of unfolded and partially folded bovine pancreatic trypsin inhibitor.
Reduced BPTI is collapsed. A pulsed field gradient NMR study of unfolded and partially folded bovine pancreatic trypsin inhibitor.
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BPTI 降低已被折叠。
DOI：
10.1002/pro.5560060919
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发表时间：
1997
期刊：
影响因子：
--
通讯作者：
Woodward,C
中科院分区：
文献类型：
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作者：
Pan,H;Barany,G;Woodward,C
利用脉冲场梯度核磁共振技术研究了牛胰胰蛋白酶抑制剂（BPTI）的非折叠变体的流体力学行为。研究的未折叠BPTI物质为pH 4.5和pH 2.5下的[R]Abu和pH 2.5下的未折叠[14 - 38] Abu。这些是通过化学合成制备的。[R] Abu是还原BPTI的模型;所有半胱氨酸残基均被α-氨基-正丁酸（Abu）取代。[14 Abu保留形成二硫键的半胱氨酸14和38，而其他半胱氨酸残基被Abu取代。在PFG实验中，扩散系数作为蛋白质浓度的函数被测量，并且D °的值-扩散系数外推到无限稀释-被确定。根据D°，流体动力学半径Rh的值由斯托克斯-爱因斯坦关系计算。在pH 4.5时，[R] Abu的Rh值显著小于无规卷曲的计算值，而在pH 2.5时，实验Rh值与无规卷曲相同。鉴于[R]Abuat pH 4.5与pH 2.5的NMR检测结构的变化（Pan H，Barbar E，Barany G，Woodward C. 1995.在还原和未折叠的牛胰腺胰蛋白酶中广泛的非随机结构（Biochemistry 34：13974 - 13981），还原的BPTI在pH 4.5下的塌陷可能与形成在序列上相隔1至3个氨基酸的侧链对的非天然疏水簇有关。还在pH 4.5下测量了[14 - 38] Abu的扩散常数，此时蛋白质部分折叠。相对于天然BPTI，部分折叠的[14 - 38] Abu的流体动力学半径的增加类似于在“熔融球”条件下测量的其他蛋白质的回转半径的增加。
Pulsed field gradient NMR was used to measure the hydrodynamic behavior of unfolded variants of bovine pancreatic trypsin inhibitor (BPTI). The unfolded BPTI species studied were [R]Abu, at pH 4.5 and pH 2.5, and unfolded [14‐38]Abuat pH 2.5. These were prepared by chemical synthesis. [R]Abuis a model for reduced BPTI; all cysteine residues are replaced by α‐amino‐n‐butyric acid (Abu). [14‐38]Aburetains cysteines 14 and 38, which form a disulfide bond, while the other cysteine residues are replaced by Abu. In the PFG experiments, the diffusion coefficient is measured as a function of protein concentration, and the value ofD°—the diffusion coefficient extrapolated to infinite dilution—is determined. From D°, a value of the hydrodynamic radius,Rh, is computed from the Stokes‐Einstein relationship. At pH 4.5, [R]Abuhas anRhvalue significantly less than the value calculated for a random coil, while at pH 2.5 the experimentalRhvalue is the same as for a random coil. In view of the changes in NMR‐detected structure of [R]Abuat pH 4.5 versus pH 2.5 (Pan H, Barbar E, Barany G, Woodward C. 1995. Extensive non‐random structure in reduced and unfolded bovine pancreatic trypsin inhibitor.Biochemistry34:13974‐13981), the collapse of reduced BPTI at pH 4.5 may be associated with the formation of non‐native hydrophobic clusters of pairs of side chains one to three amino acids apart in sequence. The diffusion constant of [14‐38]Abuwas also measured at pH 4.5, where the protein is partially folded. An increase in hydrodynamic radius of partially folded [14‐38]Aburelative to native BPTI, is similar to the increase in radius of gyration measured for other proteins under “molten globule” conditions.