Hiraga Kaori, Derbyshire Victoria, Dansereau John T, Van Roey Patrick, Belfort Marlene
Wadsworth Center, New York State Department of Health, Center for Medical Science, 150 New Scotland Avenue, Albany, NY 12208, USA.
J Mol Biol. 2005 Dec 9;354(4):916-26. doi: 10.1016/j.jmb.2005.09.088. Epub 2005 Oct 21.
Many naturally occurring inteins consist of two functionally independent domains, a protein-splicing domain and an endonuclease domain. In a previous study, a 168 amino acid residue mini-intein was generated by removal of the central endonuclease domain of the 440 residue Mycobacterium tuberculosis (Mtu) recA intein. In addition, directed evolution experiments identified a mutation, V67L, that improved the activity of the mini-intein significantly. A recent crystal structure shows that the loop connecting two beta-strands from the N-terminal and C-terminal intein subdomains of the mini-intein is disordered. The goals of the present study were to generate smaller mini-intein derivatives and to understand the basis for reversal of the splicing defect by the V67L mutation. Guided by the structural information, we generated a number of derivatives 135 to 152 residues in length, with V67 or L67. All of the new minimal inteins are functional in splicing. In vivo selection experiments for function showed that by removal of the loop region, 137 residues may be the lower limit for full protein-splicing activity. In addition, the activation effect of the V67L mutation was observed to be universal for mini-inteins longer than 137 residues. Structural and functional analyses indicate that the role of the mutation is in stabilization of the mini-intein core.
许多天然存在的内含肽由两个功能独立的结构域组成，即蛋白质剪接结构域和内切核酸酶结构域。在先前的一项研究中，通过去除440个残基的结核分枝杆菌（Mtu）recA内含肽的中央内切核酸酶结构域，产生了一个168个氨基酸残基的微型内含肽。此外，定向进化实验鉴定出一个突变V67L，该突变显著提高了微型内含肽的活性。最近的晶体结构表明，连接微型内含肽N端和C端内含肽亚结构域两条β链的环是无序的。本研究的目的是生成更小的微型内含肽衍生物，并了解V67L突变逆转剪接缺陷的基础。在结构信息的指导下，我们生成了一些长度为135至152个残基、含有V67或L67的衍生物。所有新的最小内含肽在剪接中都具有功能。功能的体内筛选实验表明，通过去除环区域，137个残基可能是完整蛋白质剪接活性的下限。此外，观察到V67L突变的激活作用对于长度超过137个残基的微型内含肽是普遍的。结构和功能分析表明，该突变的作用是稳定微型内含肽核心。
