Biogenesis, quality control, and structural dynamics of proteins as explored in living cells via site‐directed photocrosslinking

Abstract

Protein biogenesis and quality control are essential to maintaining a functional pool of proteins and involve numerous protein factors that dynamically and transiently interact with each other and with the substrate proteins in living cells. Conventional methods are hardly effective for studying dynamic, transient, and weak protein-protein interactions that occur in cells. Herein, we review how the sitedirected photocrosslinking approach, which relies on the genetic incorporation of a photoreactive unnatural amino acid into a protein of interest at selected individual amino acid residue positions and the covalent trapping of the interacting proteins upon ultraviolent irradiation, has become a highly efficient way to explore the aspects of protein contacts in living cells. For example, in the past decade, this approach has allowed the profiling of the in vivo substrate proteins of chaperones or proteases under both physiologically optimal and stressful (e.g., acidic) conditions, mapping residues located at protein interfaces, identifying new protein factors involved in the biogenesis of membrane proteins, trapping transiently formed protein complexes, and snapshotting different structural states of a protein. We anticipate that the site-directed photocrosslinking approach will play a fundamental role in dissecting the detailed mechanisms of protein biogenesis, quality control, and dynamics in the future.

Exploring proteins in living cells

“…Schematic diagrams illustrating (A) how an unnatural amino acid (e.g., p-benzoyl-L-phenylalanine [Bpa]) is genetically introduced at a selected residue position of a specific target protein A, and (B) how the nature of a protein photocrosslinked to a target protein in living cells is characterized through either immunoblotting or mass spectrometric analyses [11,15] . [16] 。 经过多年努力，这种最初在细菌中建立的方法体系，通过选择出合适的正交 tRNA 和正交氨基酰-tRNA 合成酶这两种分子， 并使它们有效表达而逐渐引入到了 真核细胞体系。具体而言，这种活细胞蛋白质非天然氨基酸标记方法已经在酵母 细胞 [17] 、体外培养的哺乳动物细胞系 [18] 、甚至是线虫这样的简单动物个体中 [19] 得到了建立 [11,15,20] [21,22] 。 Figure 2. Schematic diagram of the genes encoding the orthogonal tRNA and amino acyl-tRNA synthetase integrated at proper positions in the genome of an LY928 bacterium.…”

“…Schematic diagram of the genes encoding the orthogonal tRNA and amino acyl-tRNA synthetase integrated at proper positions in the genome of an LY928 bacterium. The schematic shows how an unnatural amino acid, in this case Bpa, is introduced via site-directed mutagenesis at the endogenous target gene in the genome [21,22] . [21,22] 将基因组上的内源性目标基因特定三联体碱基替换为 TAG(图 2) [25] 。这样的引 入了非天然氨基酸的目标蛋白质的表达特征(如，表达的时空条件和水平等)与 内源性蛋白质高度接近，这就使得实验结果更能反映真实情况。 3.4 建立了一种单块胶可分析 384 个蛋白质样品的高通量聚丙烯酰胺凝胶 电泳技术 [25] [25] 。 Figure 3.…”

“…The schematic shows how an unnatural amino acid, in this case Bpa, is introduced via site-directed mutagenesis at the endogenous target gene in the genome [21,22] . [21,22] 将基因组上的内源性目标基因特定三联体碱基替换为 TAG(图 2) [25] 。这样的引 入了非天然氨基酸的目标蛋白质的表达特征(如，表达的时空条件和水平等)与 内源性蛋白质高度接近，这就使得实验结果更能反映真实情况。 3.4 建立了一种单块胶可分析 384 个蛋白质样品的高通量聚丙烯酰胺凝胶 电泳技术 [25] [25] 。 Figure 3. A representative high-throughput polyacrylamide gel electrophoresis immunoblot image showing 384 protein samples analyzed on a single gel [25] .…”

A high‐throughput genetically directed protein crosslinking analysis reveals the physiological relevance of the ATP synthase ‘inserted’ state

“…In this regard, in vivo protein cross-linking mediated by genetically introduced photoactivatable unnatural amino acid seems to provide the most promising approach. This methodology, being initially developed by Schultz and colleagues [24,25], has been extensively employed in our laboratory [26][27][28][29][30].…”

Bifunctional Non-Canonical Amino Acids: Combining Photo-Crosslinking with Click Chemistry

“…Ideally, the covalent bond should be formed while the POI is still in its native environment, and it should affect only the POI and its binding partners. In addition, such a tag should be as small as possible so as to reduce the risk of blocking important binding sites [7].…”