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

@article{citeulike:3035683, abstract = {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 80100 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.}, address = {Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115, and Department of Molecular and Cellular Biology and Department of Chemistry and Chemical Biology, Harvard University, Cambridge, Massachusetts 02138}, author = {Zhou, Zhe and Koglin, Alexander and Wang, Yu and Mcmahon, Andrew P. and Walsh, Christopher T. }, citeulike-article-id = {3035683}, doi = {10.1021/ja802657n}, journal = {J. Am. Chem. Soc.}, keywords = {fluorescence, proteinmodification}, month = {July}, posted-at = {2008-07-23 04:50:16}, priority = {2}, title = {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}, url = {http://dx.doi.org/10.1021/ja802657n}, year = {2008} }

TY - JOUR ID - citeulike:3035683 L3 - citeulike-article-id:3035683 TI - 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 JF - J. Am. Chem. Soc. AD - Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115, and Department of Molecular and Cellular Biology and Department of Chemistry and Chemical Biology, Harvard University, Cambridge, Massachusetts 02138 N2 - 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 80100 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. KW - fluorescence KW - proteinmodification AU - Zhou, Zhe AU - Koglin, Alexander AU - Wang, Yu AU - Mcmahon, Andrew AU - Walsh, Christopher PY - 2008/07/02/ UR - http://dx.doi.org/10.1021/ja802657n ER -