An Eight Residue Fragment of an Acyl Carrier Protein Suffices for Post-Translational Introduction of Fluorescent Pantetheinyl Arms in Protein Modification in vitro and in vivo
- 1 July 2008
- journal article
- research article
- Published by American Chemical Society (ACS) in Journal of the American Chemical Society
- Vol. 130 (30), 9925-9930
- https://doi.org/10.1021/ja802657n
Abstract
Genetically encoded tags for tracking a given protein continue to be of great interest in a multitude of in vitro and in vivo contexts. Acyl carrier proteins, both free-standing and as embedded 80−100 residue domains, contain a specific serine side chain that undergoes post-translational pantetheinylation from CoASH as donor substrate. We have previously used phage display methods to select a 12 residue fragment that retains recognition for modification by the Escherichia coli phosphopantetheinyltransferase (PPTase) AcpS. In this work, we have used 15N-HSQC based NMR titration experiments of a 12-residue peptide substrate with AcpS to identify six specifically interacting residues (S3, L4, D5, M6, W9,and L11) without the formation of any notable secondary structure. Synthesis of a corresponding octapeptide containing 5 of the 6 interacting residues generated a minimal fragment capable of efficient post-translational phosphopantetheinylation. Genetic insertion of this eight residue coding sequence into the proteins sonic hedgehog and transferrin receptor enabled good in vitro and in vivo PPTase-mediated modification by a series of fluorescent CoAs, leading to a set of fluorescent proteins with a peptide tag minimally perturbant to protein folds.This publication has 20 references indexed in Scilit:
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