O'Hare P, Williams G
Marie Curie Research Institute, The Chart, Oxted, Surrey, U.K.
Biochemistry. 1992 Apr 28;31(16):4150-6. doi: 10.1021/bi00131a035.
We have overproduced and purified the carboxy-terminal transactivation domain of Vmw65 (VP16) of herpes simplex virus, and studied potential folding of the domain by 1H NMR. Two species of the acidic domain were obtained from the bacterial expression system, and we demonstrate that one of these represents read-through of the natural amber termination codon of the Vmw65 reading frame producing a larger polypeptide. Additional residues in the read-through product were identified by total amino acid analysis and by NMR. Study of the correctly terminated product by 1D NMR gave resonances which were clustered into groups around their random-coil chemical shift positions, and 2D NMR demonstrated that, even in mixed solvents containing up to 80% MeOH, there was very little evidence of secondary structure. Together these results indicate that the isolated acid domain has little if any alpha-helical content of any stable nature. We discuss these results with reference to the demonstrated activity of the acidic domain in a wide variety of polypeptide contexts.
我们已经过量生产并纯化了单纯疱疹病毒Vmw65（VP16）的羧基末端反式激活结构域，并通过1H NMR研究了该结构域的潜在折叠情况。从细菌表达系统中获得了两种酸性结构域，我们证明其中一种代表Vmw65阅读框天然琥珀终止密码子的通读，产生了一种更大的多肽。通过总氨基酸分析和NMR鉴定了通读产物中的额外残基。通过1D NMR对正确终止的产物进行研究，得到的共振峰聚集在其无规卷曲化学位移位置附近，2D NMR表明，即使在含有高达80%甲醇的混合溶剂中，也几乎没有二级结构的证据。这些结果共同表明，分离出的酸性结构域几乎没有任何稳定性质的α-螺旋含量。我们结合酸性结构域在多种多肽环境中已证实的活性来讨论这些结果。
