Institute of Biochemistry, University of Muenster, Wilhelm-Klemm-Str. 2, 48149 Münster, Germany.
Bioorg Med Chem. 2013 Jun 15;21(12):3495-503. doi: 10.1016/j.bmc.2013.03.065. Epub 2013 Apr 3.
Protein trans-splicing by split inteins holds great potential for the chemical modification and semisynthesis of proteins. However, the structural requirements of the extein sequences immediately flanking the intein are only poorly understood. This knowledge is of particular importance for protein labeling, when synthetic moieties are to be attached to the protein of interest as seamlessly as possible. Using the semisynthetic Ssp DnaB intein both in form of its wild-type sequence and its evolved M86 mutant, we systematically varied the sequence upstream of the short synthetic Int(N) fragment using both proteinogenic amino acids and unnatural building blocks. We could show for the wild-type variant that the native N-extein sequence could be reduced to the glycine residue at the (-1) position directly flanking the intein without significant loss of activity. The glycine at this position is strongly preferred over building blocks containing a phenyl group or extended alkyl chain adjacent to the scissile amide bond of the N-terminal splice junction. Despite their negative effects on the splicing yields, these unnatural substrates were well processed in the N-S acyl shift to form the respective thioesters and did not result in an increased decoupling of the asparagine cyclization step at the C-terminal splicing junction. Therefore, the transesterification step appeared to be the bottleneck of the protein splicing pathway. The fluorophore 7-hydroxycoumarinyl-4-acetic acid as a minimal N-extein was efficiently ligated to the model protein, in particular with the M86 mutant, probably because of its higher resemblance to glycine with an aliphatic c-α carbon atom at the (-1) position. This finding indicates a way for the virtually traceless labeling of proteins without inserting extra flanking residues. Due to its overall higher activity, the M86 mutant appears most promising for many protein labeling and chemical modification schemes using the split intein approach.
分裂内含肽的蛋白质反式剪接在蛋白质的化学修饰和半合成方面具有巨大的潜力。然而，紧邻内含肽的外显子序列的结构要求理解得还很差。当要将合成部分尽可能无缝地连接到感兴趣的蛋白质上时，这种知识对于蛋白质标记尤其重要。使用半合成的 Ssp DnaB 内含肽及其野生型序列和进化的 M86 突变体，我们系统地改变了短合成 Int(N) 片段上游的序列，使用了蛋白质氨基酸和非天然构建块。我们可以证明，对于野生型变体，紧邻内含肽的(-1)位置的天然 N-外显子序列可以减少到甘氨酸残基，而不会显着降低活性。该位置的甘氨酸强烈优于含有苯环或与 N 端剪接连接的酰胺键相邻的扩展烷基链的构建块。尽管它们对接合产率有负面影响，但这些非天然底物在 N-S 酰基转移中得到很好的处理，形成各自的硫酯，并且不会导致 C 端剪接连接处的天冬酰胺环化步骤增加解偶联。因此，酯交换步骤似乎是蛋白质剪接途径的瓶颈。荧光团 7-羟基香豆素基-4-乙酸作为最小的 N-外显子，与模型蛋白，特别是 M86 突变体有效地连接在一起，可能是因为其与甘氨酸的相似性更高，在(-1)位置具有脂肪族 c-α碳原子。这一发现为不插入额外的侧翼残基而对蛋白质进行几乎无痕标记指明了一条道路。由于其总体较高的活性，M86 突变体在使用分裂内含肽方法进行许多蛋白质标记和化学修饰方案中似乎最有前途。
