Perham R N
Biochem J. 1973 Jan;131(1):119-26. doi: 10.1042/bj1310119.
Several mutants of tobacco mosaic virus that contain additional lysine residues as a result of mutations in the coat protein were investigated. Mutant E66 has a lysine residue replacing asparagine at position 140 when compared with the wild-type vulgare and this lysine residue reacts readily in the intact virus with methyl picolinimidate. Mutant B13a has two new lysine residues in the coat protein, replacing a glutamine at position 9 and an asparagine at position 33, whereas mutant B13b has the single replacement of glutamine by lysine at position 9. The lysine residue at position 9 in mutants B13a and B13b also reacts readily with methyl picolinimidate in the intact virus but the lysine at position 33 in mutant B13a did not react under these conditions. However, when the isolated coat protein from mutant B13a was treated with methyl picolinimidate, the lysine residue at position 33 did become modified, showing that the loss in reactivity of this residue towards the imidoester in the intact virus is a result of the assembly of the protein subunit into the virus structure. These results are compatible with and extend previous studies on the sero-logical properties of mutants of tobacco mosaic virus and illustrate the value of methyl picolinimidate as a reagent for probing the accessibility of amino groups in proteins. When intact tobacco mosaic virus (vulgare) was treated with p-iodobenzenesulphonyl chloride, no reaction with the lysine residues at positions 33 or 68 in the virus subunit could be detected but complete modification of tyrosine-139 was achieved. This result also extends previous studies with other reagents. The usefulness of the differential reactivity of the lysine residues in tobacco mosaic virus and its mutants as a means of attaching heavy-atom labels at chemically defined positions for subsequent X-ray-diffraction analysis and the implications of these experiments for deciphering the folding of the peptide chain in the virus subunit are discussed.
研究了几种烟草花叶病毒突变体，这些突变体由于外壳蛋白发生突变而含有额外的赖氨酸残基。与野生型普通烟草相比，突变体E66在140位有一个赖氨酸残基取代了天冬酰胺，并且这个赖氨酸残基在完整病毒中能与甲基吡啶亚胺迅速反应。突变体B13a在外壳蛋白中有两个新的赖氨酸残基，分别取代了9位的谷氨酰胺和33位的天冬酰胺，而突变体B13b仅在9位有谷氨酰胺被赖氨酸取代。突变体B13a和B13b中9位的赖氨酸残基在完整病毒中也能与甲基吡啶亚胺迅速反应，但在这些条件下突变体B13a中33位的赖氨酸不发生反应。然而，当用甲基吡啶亚胺处理突变体B13a分离出的外壳蛋白时，33位的赖氨酸残基确实被修饰了，这表明该残基在完整病毒中对亚胺酯反应性的丧失是蛋白质亚基组装到病毒结构中的结果。这些结果与之前关于烟草花叶病毒突变体血清学特性的研究一致并有所扩展，同时说明了甲基吡啶亚胺作为探测蛋白质中氨基可及性试剂的价值。当用对碘苯磺酰氯处理完整的烟草花叶病毒（普通烟草）时，未检测到与病毒亚基中33位或68位赖氨酸残基的反应，但酪氨酸-139被完全修饰。这一结果也扩展了之前用其他试剂进行的研究。讨论了烟草花叶病毒及其突变体中赖氨酸残基的差异反应性作为在化学定义位置连接重原子标签以用于后续X射线衍射分析的手段的实用性，以及这些实验对解读病毒亚基中肽链折叠的意义。
