Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2014 Nov 21;9(11):2502-7.
doi: 10.1021/cb500658w. Epub 2014 Oct 6.

Designed phosphoprotein recognition in Escherichia coli

Nicholas Sawyer  1 , Brandon M Gassaway, Adrian D Haimovich, Farren J Isaacs, Jesse Rinehart, Lynne Regan
Affiliations

Designed phosphoprotein recognition in Escherichia coli

Nicholas Sawyer et al. ACS Chem Biol. .

Abstract

Protein phosphorylation is a central biological mechanism for cellular adaptation to environmental changes. Dysregulation of phosphorylation signaling is implicated in a wide variety of diseases. Thus, the ability to detect and quantify protein phosphorylation is highly desirable for both diagnostic and research applications. Here we present a general strategy for detecting phosphopeptide-protein interactions in Escherichia coli. We first redesign a model tetratricopeptide repeat (TPR) protein to recognize phosphoserine in a sequence-specific fashion and characterize the interaction with its target phosphopeptide in vitro. We then combine in vivo site-specific incorporation of phosphoserine with split mCherry assembly to observe the designed phosphopeptide-protein interaction specificity in E. coli. This in vivo strategy for detecting and characterizing phosphopeptide-protein interactions has numerous potential applications for the study of natural interactions and the design of novel ones.

PubMed Disclaimer

Figures

Figure 1
Figure 1
TRAP interaction with target phosphopeptide. (A) Model of the TRAP–phosphopeptide interaction. The entire TRAP–phosphopeptide complex is shown on the left with a zoomed-in view of the phosphopeptide binding region on the right. The TRAP protein backbone is depicted as a gray ribbon. The Cα of the phosphoserine binding pocket residues are shown as small pink spheres. Side chains for binding pocket residues (arginine at position 332 and lysine at position 334) are shown in stick representation with carbon atoms colored aqua. The phosphopeptide ME(pS)VD is shown as sticks with carbon atoms colored green. For both protein and phosphopeptide, nitrogen, oxygen, phosphorus, and sulfur atoms are colored blue, red, orange, and yellow, respectively. (B) TRAP binding to its phosphopeptide target and nonphosphopeptide analogue. Each data point shows the fraction of fluorescein-labeled peptide bound for a given TRAP concentration as measured by changes in the fluorescence anisotropy for the ME(pS)VD phosphopeptide (red) and MESVD peptide (blue). For each peptide, the solid lines show the fit of a 1:1 binding model, with dissociation constants of approximately 2 μM for the ME(pS)VD peptide and 67 μM for the MESVD peptide.
Figure 2
Figure 2
Confirmation of site-specific phosphoserine incorporation in pNAS duet/Sep-OTS coexpression system. (A) Schematic of coexpression using pNAS duet vector and Sep-OTS. Five copies of the phosphoserine-tRNA (tRNASep, purple), the phosphoserine-specific tRNA synthetase (SepRS, dark gray), and the phosphoserine-specific elongation factor (EF-Sep, light gray) are coexpressed from the B40 Sep-OTS plasmid by induction with IPTG. Two additional proteins, such as the halves of split mCherry, can be expressed from the comaintained pNAS duet vector using two additional inducers (l-(+)-arabinose and anhydrotetracycline). (B) Schematic of phosphoserine incorporation by amber suppression. White circles with 3-letter amino acid abbreviations connected by thick black lines represent the growing polypeptide whose sequence is specified by the mRNA. tRNASep charged with phosphoserine (Ser + orange star indicating phosphate group) inserts phosphoserine in response to the amber (UAG) codon. (C) Mass spectrometric confirmation of site-specific phosphoserine incorporation into the target phosphopeptide sequence. Extracted ion chromatograms are shown for the indicated mass-to-charge ratios (corresponding to the indicated peptides) for two GST fusion proteins (GST-MESVD and GST-ME(pS)VD). The indicated phosphopeptide is only detected in the GST-ME(pS)VD sample. The control serine-containing peptide is most abundant in the GST-MESVD sample.
Figure 3
Figure 3
In vivo split mCherry assembly for TRAP–phosphopeptide interactions. (A) Schematic illustration of the in vivo split mCherry protein–peptide interaction assay. On the left, the N-terminal half of mCherry (N-mCherry) is fused to a phosphopeptide (phosphate group indicated by orange star). The C-terminal half of mCherry (C-mCherry) is fused to the TRAP binding partner. When the phosphopeptide and TRAP fusion proteins are coexpressed and interact, the halves of split mCherry are brought into close proximity, assemble, and fluoresce, producing a reddish cell. On the right, neither N-mCherry nor C-mCherry is fused to anything. Although they are coexpressed in the cell, they do not interact or assemble, and no red fluorescence is observed (i.e., the cell remains beige). (B) Time-dependent increase in fluorescence for in vivo split mCherry assembly. Bars show the fluorescence intensity of E. coli cell lysates prepared at the indicated time postinduction (excitation at 587 nm and emission at 610 nm). The results for three split mCherry pairs are shown: unfused N-mCherry + unfused C-mCherry (gray), N-mCherry fused to the MESVD peptide + C-mCherry fused to the TRAP (blue), and N-mCherry fused to the ME(pS)VD peptide + C-mCherry fused to the same TRAP (red). Error bars show the standard deviation for two or three biological replicates. The inset shows the excitation and emission spectra for reassembled mCherry for the phosphopeptide-TRAP pair (lighter red for excitation and darker red for emission). The peaks observed are consistent with mCherry fluorescence despite a small shift in excitiation and emission maxima (590 and 602 nm, respectively. Such peaks are absent from the spectra for the unfused N-mCherry + C-mCherry pair (lighter gray for excitation and darker gray for emission). (C) Flow cytometry measurements of E. coli cells containing different split mCherry pairs. Fluorescence histograms are shown for three split mCherry pairs: N-mCherry + C-mCherry (gray), N-mCherry fused to the MESVD peptide + C-mCherry fused to the TRAP (blue), and N-mCherry fused to the ME(pS)VD peptide + C-mCherry fused to the same TRAP (red). The mean fluorescence is 0.653 for N-mCherry + C-mCherry, 10.8 for N-mCherry fused to the MESVD peptide + C-mCherry fused to the TRAP, and 22.1 for N-mCherry fused to the ME(pS)VD peptide + C-mCherry fused to the same TRAP.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Khoury G. A.; Baliban R. C.; Floudas C. A. (2011) Proteome-wide post-translational modification statistics: frequency analysis and curation of the swiss-prot database. Sci. Rep. 1, 90. - PMC - PubMed
    1. Canagarajah B. J.; Khokhlatchev A.; Cobb M. H.; Goldsmith E. J. (1997) Activation mechanism of the MAP kinase ERK2 by dual phosphorylation. Cell 90, 859–869. - PubMed
    1. Stoothoff W. H.; Johnson G. V. W. (2005) Tau phosphorylation: physiological and pathological consequences. Biochim. Biophys. Acta 1739, 280–297. - PubMed
    1. Dhillon A. S.; Hagan S.; Rath O.; Kolch W. (2007) MAP kinase signalling pathways in cancer. Oncogene 26, 3279–3290. - PubMed
    1. Nairn A. C.; Detre J. A.; Casnellie J. E.; Greengard P. (1982) Serum antibodies that distinguish between the phospho- and dephospho-forms of a phosphoprotein. Nature 299, 734–736. - PubMed

Publication types

Substances