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.
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 (15)N-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.
Zhou
Zhe
Z
Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School,Boston, Massachusetts 02115, USA.
Koglin
Alexander
A
Wang
Yu
Y
McMahon
Andrew P
AP
Walsh
Christopher T
CT
D000213:Acyl Carrier Protein / Q000737:chemistry* / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry* / Q000378:metabolism; D003065:Coenzyme A / Q000737:chemistry* / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000378:metabolism; D005455:Fluorescent Antibody Technique; D005456:Fluorescent Dyes / Q000737:chemistry*; D049452:Green Fluorescent Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D053823:Hedgehog Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods; D009842:Oligopeptides / Q000737:chemistry* / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D011990:Receptors, Transferrin / Q000737:chemistry / Q000235:genetics; D013379:Substrate Specificity; D014166:Transferases / Q000737:chemistry* / Q000378:metabolism; D017855:Transferases (Other Substituted Phosphate Groups) / Q000737:chemistry* / Q000378:metabolism
D000213:Acyl Carrier Protein; D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D005456:Fluorescent Dyes; D053823:Hedgehog Proteins; D009842:Oligopeptides; D011990:Receptors, Transferrin; C082662:phosphopantetheinyl transferase; C073125:acpS protein, E coli; D049452:Green Fluorescent Proteins; D014166:Transferases; D017855:Transferases (Other Substituted Phosphate Groups); D003065:Coenzyme A