Tsuboi M, Overman S A, Thomas G J
Division of Cell Biology and Biophysics, School of Biological Sciences, University of Missouri-Kansas City, Missouri 64110, USA.
Biochemistry. 1996 Aug 13;35(32):10403-10. doi: 10.1021/bi9527707.
The Ff filamentous virus, which includes the closely related strains fd, fl and M13, serves as a model for membrane protein assembly and is employed extensively as a cloning vector and vehicle for peptide display. The threadlike virion (approximately 6 x 880 nm) comprises a single-stranded DNA genome sheathed by approximately 2700 copies of a 50-residue alpha-helical subunit, the product of viral gene VIII. The pVIII subunit contains a single tryptophan residue (tryptophan-26) which is essential for assembly. We have employed polarized Raman microspectroscopy to determine the orientation of tryptophan-26 in pVIII subunits of oriented fd fibers. The present application is based upon the transfer of tryptophan Raman tensors from a recent study of N-acetyl-L-tryptophan single crystals [Tsuboi et al. (1996) J. Mol. Struct. 379, 43-50]. The polarized Raman spectra of fd indicate that the plane of the indole ring in each pVIII subunit is close to parallel to the virion axis. In this orientation, the line connecting indole ring atoms N1 and C2 is nearly perpendicular to the virion axis, while the indole pseudo-2-fold axis (a line connecting atom C2 to the midpoint of the C5-C6 bond) is approximately 36 degrees from the virion axis. We have used the present results in combination with preferred tryptophan side-chain torsions [chi 1 (C3-C beta-C alpha-N) and chi 2.1 (C2-C3-C beta-C alpha)] in other proteins and a previously determined experimental value of chi 2.1 in fd [Aubrey. K. L., & Thomas, G. J., Jr. (1991) Biophys, J. 60, 1337-1349] to propose a detailed molecular model for the orientation of the tryptophan-26 side chain in the native virus.
Ff丝状病毒，包括密切相关的fd、fl和M13菌株，是膜蛋白组装的模型，被广泛用作克隆载体和肽展示载体。丝状病毒粒子（约6×880纳米）由单链DNA基因组组成，该基因组被约2700个50个残基的α-螺旋亚基包裹，该亚基是病毒基因VIII的产物。pVIII亚基包含一个单一的色氨酸残基（色氨酸-26），这对组装至关重要。我们利用偏振拉曼光谱法来确定fd纤维中pVIII亚基上色氨酸-26的取向。本应用基于最近对N-乙酰-L-色氨酸单晶的研究中色氨酸拉曼张量的转移[Tsuboi等人（1996年）《分子结构杂志》379，43-50]。fd的偏振拉曼光谱表明，每个pVIII亚基中吲哚环的平面接近与病毒粒子轴平行。在这个取向下，连接吲哚环原子N1和C2的线几乎垂直于病毒粒子轴，而吲哚假二重轴（连接原子C2到C5-C6键中点的线）与病毒粒子轴大约成36度角。我们将目前的结果与其他蛋白质中色氨酸侧链的优选扭转[χ1（C3-Cβ-Cα-N）和χ2.1（C2-C3-Cβ-Cα）]以及先前确定的fd中χ2.1的实验值[Aubrey. K. L.，& Thomas, G. J., Jr.（1991年）《生物物理学杂志》60，1337-1349]相结合，提出了天然病毒中色氨酸-26侧链取向的详细分子模型。
