Khan A R, Williams K A, Boggs J M, Deber C M
Division of Biochemistry Research, Hospital for Sick Children, Toronto, Ontario, Canada.
Biochemistry. 1995 Sep 26;34(38):12388-97. doi: 10.1021/bi00038a036.
The filamentous bacteriophage major coat protein occurs as a membrane-spanning assembly intermediate prior to incorporation into the lipid-free virion. To gain insight into how this small, multifunctional protein is able to be stably incorporated into both of these distinct environments, the reactive sulfhydryl group of IKe and M13 coat protein Cys mutants was exploited to probe the mobility and environment of this residue at several loci within the hydrophobic domain of these proteins. IKe mutants P30C, G39C, and G39C-V36A and M13 mutant Y24C-V31A, each previously obtained from randomized mutagenesis, were characterized in the intact virion, the intermediate spheroidal S-form, and in membrane-mimetic sodium dodecyl sulfate (SDS) micelles. The accessibility of the Cys sulfhydryl in the virion was examined by reaction with [14C]iodoacetamide (14C-IAN) and other alkylating agents. The IKe mutants G39C and G39C-V36A were found to be the most reactive with 14C-IAN and thus the most accessible, although this accessibility was subject to strict steric constraints since only the smallest sulfhydryl-specific alkylating agents were able to modify the Cys39 locus. The spin probe proxyliodoacetamide (PIAN) was used to characterize Cys side chain mobility by electron paramagnetic resonance (EPR) spectroscopy. The M13 mutant Y24C-V31A Cys side chain in the phage was observed to be the most mobile, with slightly less mobility for IKe mutant P30C and considerably less for G39C mutants. The SDS micelle-bound forms of the Cys mutants all exhibited enhanced side chain mobility compared to the virion form, with the extent of mobility dependent upon the specific location of the Cys residue. EPR and fluorescence quenching results show that the Cys side chain in the Y24C-V31A S-form is largely immobilized and inaccessible in comparison to the virion and micelle-solubilized forms. The overall results are interpreted in terms of the structural changes accompanying disassembly and insertion of the coat protein into the Escherichia coli inner membrane.
丝状噬菌体主要衣壳蛋白在整合到无脂病毒粒子之前，以跨膜组装中间体的形式存在。为深入了解这种小的多功能蛋白如何能够稳定地整合到这两种不同的环境中，利用IKe和M13衣壳蛋白半胱氨酸突变体的反应性巯基来探测该残基在这些蛋白疏水结构域内几个位点的流动性和环境。IKe突变体P30C、G39C和G39C-V36A以及M13突变体Y24C-V31A，每个都是先前通过随机诱变获得的，在完整病毒粒子、中间球状S型以及膜模拟十二烷基硫酸钠（SDS）胶束中进行了表征。通过与[14C]碘乙酰胺（14C-IAN）和其他烷基化剂反应，检测病毒粒子中半胱氨酸巯基的可及性。发现IKe突变体G39C和G39C-V36A与十四碳碘乙酰胺反应性最强，因此最易接近，不过这种可及性受到严格的空间限制，因为只有最小的巯基特异性烷基化剂能够修饰Cys39位点。自旋探针碘代丙酰胺（PIAN）用于通过电子顺磁共振（EPR）光谱表征半胱氨酸侧链的流动性。观察到噬菌体中的M13突变体Y24C-VI31A半胱氨酸侧链流动性最强，IKe突变体P30C的流动性稍低，G39C突变体的流动性则低得多。与病毒粒子形式相比，半胱氨酸突变体的SDS胶束结合形式均表现出增强的侧链流动性，流动性程度取决于半胱氨酸残基的具体位置。EPR和荧光猝灭结果表明，与病毒粒子和胶束溶解形式相比，Y24C-V31A S型中的半胱氨酸侧链在很大程度上固定不动且难以接近。根据衣壳蛋白拆卸和插入大肠杆菌内膜时伴随的结构变化对总体结果进行了解释。
