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Reversible labeling of native and fusion-protein motifs.

Kosa NM, Haushalter RW, Smith AR, Burkart MD - Nat. Methods (2012)

Bottom Line: The reversible covalent attachment of chemical probes to proteins has long been sought as a means to visualize and manipulate proteins.Here we demonstrate the full reversibility of post-translational custom pantetheine modification of Escherichia coli acyl carrier protein for visualization and functional studies.We use this iterative enzymatic methodology in vitro to reversibly label acyl carrier protein variants and apply these tools to NMR structural studies of protein-substrate interactions.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry and Biochemistry, University of California, San Diego, La Jolla, California, USA.

ABSTRACT
The reversible covalent attachment of chemical probes to proteins has long been sought as a means to visualize and manipulate proteins. Here we demonstrate the full reversibility of post-translational custom pantetheine modification of Escherichia coli acyl carrier protein for visualization and functional studies. We use this iterative enzymatic methodology in vitro to reversibly label acyl carrier protein variants and apply these tools to NMR structural studies of protein-substrate interactions.

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Related in: MedlinePlus

Reversible labeling of E. coli ACPRecombinant ACP-15N is isolated in the holo- state (top, yellow). ACP-15N is prepared for covalent labeling by treatment with AcpH to generate exclusively apo-ACP-15N (red). Protein purity and modification homogeneity is confirmed by 2D-NMR (top right). Labeling with acyl pantetheine analogs to the crypto-ACP-15N (blue), or labeled form, proceeds via PPTase and mCoA modification that is analyzed by NMR (bottom right, top left). Modification is quantitatively reversed by AcpH, returning labeled proteins to the apo- form.
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Figure 1: Reversible labeling of E. coli ACPRecombinant ACP-15N is isolated in the holo- state (top, yellow). ACP-15N is prepared for covalent labeling by treatment with AcpH to generate exclusively apo-ACP-15N (red). Protein purity and modification homogeneity is confirmed by 2D-NMR (top right). Labeling with acyl pantetheine analogs to the crypto-ACP-15N (blue), or labeled form, proceeds via PPTase and mCoA modification that is analyzed by NMR (bottom right, top left). Modification is quantitatively reversed by AcpH, returning labeled proteins to the apo- form.

Mentions: Post-translational protein modification is important for adding functions to proteins that can be exploited for therapeutics1, protein engineering2, affinity design3,4, and enzyme immobilization5, among other applications6. Acyl carrier protein (ACP) labeling with 4’-phosphopantetheine (ppant), conjugated to a tag of choice by its transferase (PPTase) represents one of the most flexible covalent protein labeling methods, as illustrated by the application of ACP tagged peptides7, for bio-gel formation8, and ACP-dependent protein immobilization9. Labeling ACP and ACP fusion proteins with ppant analogs is also successfully leveraged for visualization10,11, isolation12, functional13, and structural14,15 studies of carrier protein-dependent biosynthetic enzymes16. Yet further advancement of these tools is hampered by an inability to easily reverse ppant attachment. Indeed, naturally occurring ACPs, often isolated in holo- form (including a native ppant modification), cannot be further modified directly with another ppant tag. To overcome these difficulties, we use AcpH (acyl carrier protein hydrolase), a phosphodiesterase from Pseudomonas aeruginosa17 and Sfp, a PPTase from Bacillus subtilis18 to swap different ppant-conjugated small molecules on free ACP and ACP fusion proteins (Fig. 1). This reversible tagging system offers the ability to connect synthetic and biological chemistry with ease and provides uniformly labeled, high quality ACP and ACP fusion proteins, as demonstrated here through fluorescent labeling and solution-phase protein NMR.


Reversible labeling of native and fusion-protein motifs.

Kosa NM, Haushalter RW, Smith AR, Burkart MD - Nat. Methods (2012)

Reversible labeling of E. coli ACPRecombinant ACP-15N is isolated in the holo- state (top, yellow). ACP-15N is prepared for covalent labeling by treatment with AcpH to generate exclusively apo-ACP-15N (red). Protein purity and modification homogeneity is confirmed by 2D-NMR (top right). Labeling with acyl pantetheine analogs to the crypto-ACP-15N (blue), or labeled form, proceeds via PPTase and mCoA modification that is analyzed by NMR (bottom right, top left). Modification is quantitatively reversed by AcpH, returning labeled proteins to the apo- form.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4128096&req=5

Figure 1: Reversible labeling of E. coli ACPRecombinant ACP-15N is isolated in the holo- state (top, yellow). ACP-15N is prepared for covalent labeling by treatment with AcpH to generate exclusively apo-ACP-15N (red). Protein purity and modification homogeneity is confirmed by 2D-NMR (top right). Labeling with acyl pantetheine analogs to the crypto-ACP-15N (blue), or labeled form, proceeds via PPTase and mCoA modification that is analyzed by NMR (bottom right, top left). Modification is quantitatively reversed by AcpH, returning labeled proteins to the apo- form.
Mentions: Post-translational protein modification is important for adding functions to proteins that can be exploited for therapeutics1, protein engineering2, affinity design3,4, and enzyme immobilization5, among other applications6. Acyl carrier protein (ACP) labeling with 4’-phosphopantetheine (ppant), conjugated to a tag of choice by its transferase (PPTase) represents one of the most flexible covalent protein labeling methods, as illustrated by the application of ACP tagged peptides7, for bio-gel formation8, and ACP-dependent protein immobilization9. Labeling ACP and ACP fusion proteins with ppant analogs is also successfully leveraged for visualization10,11, isolation12, functional13, and structural14,15 studies of carrier protein-dependent biosynthetic enzymes16. Yet further advancement of these tools is hampered by an inability to easily reverse ppant attachment. Indeed, naturally occurring ACPs, often isolated in holo- form (including a native ppant modification), cannot be further modified directly with another ppant tag. To overcome these difficulties, we use AcpH (acyl carrier protein hydrolase), a phosphodiesterase from Pseudomonas aeruginosa17 and Sfp, a PPTase from Bacillus subtilis18 to swap different ppant-conjugated small molecules on free ACP and ACP fusion proteins (Fig. 1). This reversible tagging system offers the ability to connect synthetic and biological chemistry with ease and provides uniformly labeled, high quality ACP and ACP fusion proteins, as demonstrated here through fluorescent labeling and solution-phase protein NMR.

Bottom Line: The reversible covalent attachment of chemical probes to proteins has long been sought as a means to visualize and manipulate proteins.Here we demonstrate the full reversibility of post-translational custom pantetheine modification of Escherichia coli acyl carrier protein for visualization and functional studies.We use this iterative enzymatic methodology in vitro to reversibly label acyl carrier protein variants and apply these tools to NMR structural studies of protein-substrate interactions.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry and Biochemistry, University of California, San Diego, La Jolla, California, USA.

ABSTRACT
The reversible covalent attachment of chemical probes to proteins has long been sought as a means to visualize and manipulate proteins. Here we demonstrate the full reversibility of post-translational custom pantetheine modification of Escherichia coli acyl carrier protein for visualization and functional studies. We use this iterative enzymatic methodology in vitro to reversibly label acyl carrier protein variants and apply these tools to NMR structural studies of protein-substrate interactions.

Show MeSH
Related in: MedlinePlus