Genetically Encoded Short Peptide Tags for Orthogonal Protein Labeling by Sfp and AcpS Phosphopantetheinyl Transferases

Abstract

Short peptide tags S6 and A1, each 12 residues in length, were identified from a phage-displayed peptide library as efficient substrates for site-specific protein labeling catalyzed by Sfp and AcpS phosphopantetheinyl transferases (PPTases), respectively. S6 and A1 tags were selected for useful levels of orthogonality in reactivities with the PPTases: the catalytic efficiency, kcat/Km of Sfp-catalyzed S6 serine phosphopantetheinylation was 442-fold greater than that for AcpS. Conversely, the kcat/Km of AcpS-catalyzed A1 labeling was 30-fold higher than that for Sfp-catalyzed A1 labeling. S6 and A1 peptide tags can be fused to N- or C-termini of proteins for orthogonal labeling of target proteins in cell lysates or on live cell surfaces. The development of the orthogonal S6 and A1 tags represents a significant enhancement of PPTase-catalyzed protein labeling, allowing tandem or iterative covalent attachment of small molecules of diverse structures to the target proteins with high efficiency and specificity.

Selective translocation of intracellular Smoothened to the primary cilium in response to Hedgehog pathway modulation

“…Further, the ''built-in'' spatial resolution of the system, a property of the cell exclusion of CoA substrates, enabled us to study the mechanisms of Smo trafficking distinguishing cell membrane populations from intracellular pools. Future developments such as orthogonal labeling with two different PPTases, Sfp, and AcpS, will enable the investigation of multiple proteins simultaneously (32) and the development of cell permeable substrates will expand this important methodology enabling biomedical researchers to extend molecular imaging studies.…”

“…AcpS labeling reactions were performed as described in ref. 32. Fluorescent images were collected after fixing samples, using a Zeiss LSM510 META on an inverted microscope, with a 63ϫ (1.4 NA) oil-immersion objective lens, using 4 laser lines (405, 488, 543, 633 nm).…”

“…AcpS enzyme and CoA substrates were added to the culture medium to label A1::Smo::GFP in living cells. Because the high, negative charge of the CoA substrates prevents their penetration through the cell membrane, only A1::Smo::GFP on the cell surface can be labeled by this approach (32,33). Thus, A1::Smo::GFP can be distinguished in cell membrane and intracellular pools.…”

“…However, the large mass of the FP component and their restricted spectra limit their application. Site specific posttranslational labeling is an emerging complementary technology (re- (32,33). PPTase labeling enables membrane proteins to be tracked with ''built-in'' high temporal and spatial resolution, with a minimal shift in molecular mass and a growing library of bright, photostable fluorophores that act across the visible spectrum.…”

Chemical tags: Applications in live cell fluorescence imaging

“…The covalent linkage of a 4 0 -phosphopantetheine-probe to a serin residue in ACP is formed by the enzyme phosphopantetheine transferase (PPTase). However, the charged substrate has difficulty crossing cell membranes, which is why the ACP-tag is only suited for labeling cell surface proteins [13][14][15].…”