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• W1990373397 abstract "Expressed protein ligation (EPL) is a semisynthetic technique in which a recombinant protein thioester, generated by thiolysis of an intein fusion protein, is reacted with a synthetic or recombinant peptide with an N-terminal cysteine to produce a native peptide bond. This method has been used extensively for the incorporation of biophysical probes, unnatural amino acids, and post-translational modifications in proteins. In the 10 years since this technique was developed, the applications of EPL to studying protein structure and function have grown ever more sophisticated. In this Account, we review the use of EPL in selected systems in which substantial mechanistic insights have recently been gained through the use of the semisynthetic protein derivatives. EPL has been used in many studies to unravel the complexity of signaling networks and subcellular trafficking. Herein, we highlight this application to two different systems. First, we describe how phosphorylated or otherwise modified proteins in the TGF-beta signaling network were prepared and how they were applied to understanding the complexities of this pathway, from receptor activation to nuclear import. Second, Rab-GTPases are multiply modified with lipid derivatives, and EPL-based techniques were used to incorporate these modifications, allowing for the elucidation of the biophysical basis of membrane association and dissociation. We also review the use of EPL to understand the biology of two other systems, the potassium channel KcsA and histones. EPL was used to incorporate d-alanine and an amide-to-ester backbone modification in the selectivity filter of the KcsA potassium channel, providing insight into the mechanism of selectivity in ion conduction. In the case of histones, which are among the most heavily post-translationally modified proteins, the modifications play a key role in the regulation of gene transcription and chromatin structure. We describe how native chemical ligation and EPL were used to generate acetylated, phosphorylated, methylated, and ubiquitylated histones and how these modified histones were used to interrogate chromatin biology. Collectively, these studies demonstrate the utility of EPL in protein science. These techniques and concepts are applicable to many other systems, and ongoing advances promise to extend this semisynthetic technique to increasingly complex biological problems." @default.
• W1990373397 created "2016-06-24" @default.
• W1990373397 creator A5031060352 @default.
• W1990373397 creator A5081788616 @default.
• W1990373397 date "2008-10-22" @default.
• W1990373397 modified "2023-10-16" @default.
• W1990373397 title "Expressed Protein Ligation (EPL) in the Study of Signal Transduction, Ion Conduction, And Chromatin Biology" @default.
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• W1990373397 doi "https://doi.org/10.1021/ar800129c" @default.
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