DISULFIDE BOND ISOMERIZATION IN BPTI AND BPTI(G36S) - AN NMR-STUDY OF CORRELATED MOBILITY IN PROTEINS
DISULFIDE BOND ISOMERIZATION IN BPTI AND BPTI(G36S) - AN NMR-STUDY OF CORRELATED MOBILITY IN PROTEINS
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DOI：
10.1021/bi00065a008
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发表时间：
1993-04-13
期刊：
影响因子：
2.9
通讯作者：
WUTHRICH, K
中科院分区：
文献类型：
--
作者：
OTTING, G;LIEPINSH, E;WUTHRICH, K
在碱性胰蛋白酶抑制剂（BPTI）和Gly 36被Ser取代的突变蛋白BPTI（G36 S）的H-1核磁共振（NMR）谱中观察到两种构象异构体。丰度较低的异构体与主要构象的不同之处在于Cys 14-Cys 38二硫键的不同手性。在BPTI中，次要构象异构体的数量从4 ℃时的约1.5%增加到68 ℃时的8%。在BPTI（G36 S），人口的次要构象是约15%的总蛋白质，使详细的结构研究在技术上是可行的;在较高的温度下的次要构象的人口增加的趋势也观察到这种突变蛋白。使用均匀富集N-15的蛋白质样品，在4-68 ℃的温度范围内测量两种构象之间交换的活化参数。在室温以下的二硫键翻转的交换速率遵循Arrhenius型温度依赖性，在两种蛋白质中具有负活化熵。在较高温度下，交换速率由不同的活化参数集控制，其类似于Tyr 35围绕C（β）-C（γ）键的环翻转。虽然平衡焓和熵在很大程度上与温度无关，但活化熵在较高温度下改变符号并为正。这些结果表明，在室温以上，二硫键翻转与Tyr 35的环翻转相同的蛋白质结构波动偶联。
Two conformational isomers were observed in the H-1 nuclear magnetic resonance (NMR) spectra of the basic pancreatic trypsin inhibitor (BPTI) and of a mutant protein with Gly 36 replaced by Ser, BPTI(G36S). The less abundant isomer differs from the major conformation by different chirality of the Cys 14-Cys 38 disulfide bond. In BPTI, the population of the minor conformer increases from about 1.5% at 4-degrees-C to 8% at 68-degrees-C. In BPTI(G36S), the population of the minor conformation is about 15% of the total protein, so that a detailed structural study was technically feasible; a trend toward increasing population of the minor conformer at higher temperatures was observed also for this mutant protein. The activation parameters for the exchange between the two conformations were measured in the temperature range 4-68-degrees-C, using uniformly N-15-enriched protein samples. Below room temperature the exchange rate of the disulfide flip follows an Arrhenius-type temperature dependence, with negative activation entropy in both proteins. At higher temperatures the exchange rates are governed by a different set of activation parameters, which are similar to those for the ring flips of Tyr 35 about the C(beta)-C(gamma) bond. Although the equilibrium enthalpy and entropy were found to be largely temperature independent, the activation entropy changes sign and is positive at higher temperatures. These results suggest that, above room temperature, the disulfide flips are coupled to the same protein structure fluctuations as the ring flips of Tyr 35.