Overman S A, Thomas G J
Division of Cell Biology and Biophysics, School of Biological Sciences, University of Missouri-Kansas City 64110, USA.
Biochemistry. 1995 Apr 25;34(16):5440-51. doi: 10.1021/bi00016a015.
Site-specific isotope substitutions in the coat protein (pVIII) of the filamentous bacterial virus Ff (fd, fl, M13) have been employed to advance vibrational band assignments and facilitate structural interpretation of the Raman spectrum. We report spectra of phage fd assembled in vivo from pVIII subunits incorporating either deuteriophenylalanine (Fd5), deuteriotryptophan (Wd5), or deuteriotyrosine (Yd4) residues with labeled ring sites. The deuterated aromatics were introduced into fd individually and in combination. On the basis of observed isotope shifts, definitive assignments have been developed for all prominent Raman bands diagnostic of the pVIII aromatic residues (F11, F42, F45, W26, Y21, Y24). The present study constitutes the first direct experimental determination of Raman fingerprints of tyrosine and phenylalanine side chains within hydrophobic alpha-helical domains and yields unexpected results. Importantly, neither Y21 nor Y24 of pVIII exhibits the "canonical" Fermi doublet expected in the 820-860 cm-1 interval of the Raman spectrum. Instead, each tyrosine exhibits a single band near 853 cm-1. Since the application of denaturing conditions is sufficient to generate in fd an apparent Fermi doublet, it is concluded that the anomalous singlet is intrinsic to tyrosine environments in the native virion assembly. In addition, the Raman results clearly demonstrate an interdependence of the environments of aromatic side chains in virion subunits. We show that the results on fd isotopomers are also confirmed by Raman spectroscopy of Ff virions incorporating the tyrosine mutations Y21M, Y24M, and Y21F/Y24S. The Raman marker bands identified for pVIII aromatics modify and extend Raman correlations proposed previously for proteins. The unusual environments detected for aromatic residues in the mature Ff assembly are discussed in relation to recently proposed models for filamentous virion architecture.
丝状细菌病毒Ff（fd、fl、M13）衣壳蛋白（pVIII）中的位点特异性同位素取代已被用于推进振动带归属，并有助于对拉曼光谱进行结构解释。我们报告了在体内由掺入氘代苯丙氨酸（Fd5）、氘代色氨酸（Wd5）或氘代酪氨酸（Yd4）残基且环位点被标记的pVIII亚基组装而成的噬菌体fd的光谱。将氘代芳烃单独或组合引入fd。基于观察到的同位素位移，已对所有诊断pVIII芳香族残基（F11、F42、F45、W26、Y21、Y24）的突出拉曼带进行了明确归属。本研究首次直接实验测定了疏水α-螺旋结构域内酪氨酸和苯丙氨酸侧链的拉曼指纹图谱，并得出了意想不到的结果。重要的是，pVIII的Y21和Y24在拉曼光谱820 - 860 cm-1区间均未表现出预期的“典型”费米双峰。相反，每个酪氨酸在853 cm-1附近表现出一条单带。由于应用变性条件足以在fd中产生明显的费米双峰，因此得出结论，异常单峰是天然病毒体组装中酪氨酸环境所固有的。此外，拉曼结果清楚地表明病毒体亚基中芳香族侧链环境之间存在相互依赖性。我们表明，掺入酪氨酸突变Y21M、Y24M和Y21F/Y24S的Ff病毒体的拉曼光谱也证实了关于fd同位素异构体的结果。为pVIII芳香族物质鉴定出的拉曼标记带修改并扩展了先前针对蛋白质提出的拉曼相关性。结合最近提出的丝状病毒体结构模型，讨论了在成熟Ff组装体中检测到的芳香族残基的异常环境。
