Tsuboi M, Ushizawa K, Nakamura K, Benevides J M, Overman S A, Thomas G J
Division of Cell Biology and Biophysics, School of Biological Sciences, University of Missouri-Kansas City, Kansas City, Missouri 64110, USA.
Biochemistry. 2001 Feb 6;40(5):1238-47. doi: 10.1021/bi001936n.
The capsid of filamentous virus Ff is assembled from approximately 2750 copies of a 50-residue alpha-helical subunit, the two tyrosines of which (Tyr 21 and Tyr 24) are located within a hydrophobic sequence that constitutes the subunit interface. We have determined the side chain orientations of Tyr 21 and Tyr 24 by polarized Raman microspectroscopy of oriented Ff fibers, utilizing a novel experimental approach that combines site-specific mutation and residue-specific deuteration of capsid subunits. The polarized Raman signature of Tyr 21 was obtained by incorporating C(delta 1),C(delta 2),C(epsilon 1),C(epsilon 2)-tetradeuteriotyrosine at position 21 in an Ff mutant in which Tyr 24 is replaced with methionine. Similarly, the polarized Raman signature of Tyr 24 was obtained by incorporating C(delta 1),C(delta 2),C(epsilon 1),C(epsilon 2)-tetradeuteriotyrosine at position 24 in the analogous Tyr 21 --> Met mutant. Polarizations of the corresponding C-D stretching bands in the 2200-2400 cm(-1) interval of the Raman spectrum were measured and interpreted using tensors transferred from a polarized Raman analysis of L-tyrosine-2,3,5,6-d(4) single crystals. Polarized Raman analysis was extended to the bands of Ff near 642 and 855 cm(-1), which originate from vibrational modes of the tyrosine phenolic ring. The results indicate the following: (i) For both Tyr 21 and Tyr 24, the phenolic 2-fold axis (C(1)-C(4) line) is inclined at 41 +/- 5 degrees from the virion axis and the normal to the plane of the phenolic ring is inclined at 71 +/- 5 degrees from the virion axis; (ii) the mutation of Tyr 24, but not the mutation of Tyr 21, perturbs Raman markers of the subunit tryptophan (Trp 26), suggesting interdependence of Tyr 24 and Trp 26 orientations in native Ff; and (iii) polarization anisotropies observed for Raman markers of Ff DNA bases are unperturbed by mutation of either Tyr 21 or Tyr 24, indicating that nonrandom base orientations of packaged Ff DNA are independent of the mutation of either Tyr 21 or Tyr 24. A molecular model consistent with these findings is proposed.
丝状病毒Ff的衣壳由大约2750个50个残基的α-螺旋亚基组装而成，其中两个酪氨酸（Tyr 21和Tyr 24）位于构成亚基界面的疏水序列内。我们通过对定向Ff纤维进行偏振拉曼显微光谱分析，利用一种结合衣壳亚基的位点特异性突变和残基特异性氘代的新实验方法，确定了Tyr 21和Tyr 24的侧链取向。Tyr 21的偏振拉曼特征是通过在Tyr 24被甲硫氨酸取代的Ff突变体的第21位掺入C（δ1）、C（δ2）、C（ε1）、C（ε2）-四氘代酪氨酸获得的。同样，Tyr 24的偏振拉曼特征是通过在类似的Tyr 21→Met突变体的第24位掺入C（δ1）、C（δ2）、C（ε1）、C（ε2）-四氘代酪氨酸获得的。测量并使用从L-酪氨酸-2,3,5,6-d（4）单晶的偏振拉曼分析转移的张量解释拉曼光谱在2200 - 2400 cm（-1）区间内相应C-D伸缩带的偏振。偏振拉曼分析扩展到Ff在642和855 cm（-1）附近的谱带，这些谱带源于酪氨酸酚环的振动模式。结果表明：（i）对于Tyr 21和Tyr 24，酚的二重轴（C（1）-C（4）线）与病毒粒子轴倾斜41±5度，酚环平面的法线与病毒粒子轴倾斜71±5度；（ii）Tyr 24的突变而非Tyr 21的突变扰乱了亚基色氨酸（Trp 26）的拉曼标记，表明在天然Ff中Tyr 24和Trp 26取向相互依赖；（iii）Ff DNA碱基的拉曼标记观察到的偏振各向异性不受Tyr 21或Tyr 24突变的影响，表明包装的Ff DNA的非随机碱基取向独立于Tyr 21或Tyr 24的突变。提出了一个与这些发现一致的分子模型。
