Genetically encoded fluorescent tags for visualization of proteins in living cells add six to several hundred amino acids to the protein of interest. While suitable for most proteins, common tags easily match and exceed the size of microproteins of 60 amino acids or less. The added molecular weight and structure of such fluorescent tag may thus significantly affect in vivo biophysical and biochemical properties of microproteins. Here, we develop single-residue terminal labeling (STELLA) tags that introduce a single noncanonical amino acid either at the N- or C-terminus of a protein or microprotein of interest for subsequent specific fluorescent labeling. Efficient terminal noncanonical amino acid mutagenesis is achieved using a precursor tag that is tracelessly cleaved. Subsequent selective bioorthogonal reaction with a cell-permeable organic dye enables live cell imaging of microproteins with minimal perturbation of their native sequence. The use of terminal residues for labeling provides a universally applicable and easily scalable strategy, which avoids alteration of the core sequence of the microprotein.

中文翻译：

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用于微蛋白活细胞荧光成像的通用单残基末端标记

用于可视化活细胞中蛋白质的基因编码荧光标签为感兴趣的蛋白质添加了六到数百个氨基酸。虽然适用于大多数蛋白质，但常见标签很容易匹配并超过 60 个或更少氨基酸的微生物蛋白质的大小。因此，这种荧光标签的添加分子量和结构可能会显着影响微生物蛋白质的体内生物物理和生化特性。在这里，我们开发了单残基末端标记 (STELLA) 标签，该标签在感兴趣的蛋白质或微生物蛋白的 N 或 C 末端引入单个非规范氨基酸，用于随后的特定荧光标记。使用无痕切割的前体标签实现有效的末端非经典氨基酸诱变。随后与细胞渗透性有机染料的选择性生物正交反应能够对微生物蛋白进行活细胞成像，同时对其天然序列的干扰最小。使用末端残基进行标记提供了一种普遍适用且易于扩展的策略，可避免改变微生物蛋白的核心序列。