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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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Gel Detection of Reversible ACP Labeling(a) Analysis of rhodamine-labeled crypto-ACP confirms AcpH’s ablity to remove rhodamine-pantetheine (separate lane of same gel indicated by demarcation). (b) Reversibly labeling fusion-ACPs: MBP-PaACP, GFP-ACP, and Luciferase-ACP. Apo- ACP fusions are labeled with rhodamine-CoA (mCoA) and Sfp, and subsequently unlabeled with AcpH (separate gels indicated by demarcation). (c) Acyl-pantetheine analogs were installed on ACP-15N used for NMR analysis. ACP standards for apo- and holo- allow labeling evaluation. Initial ACP-15N apo and holo mixture is readily converted to full apo- with AcpH. “One-Pot” Sfp and AcpH methodology allows conversion of one protein sample to octanoyl-, butanoyl-13C4-, and octanoyl-8-13C1-ACP-15N. Full length gels are presented in Supplementary Information (Supplementary Fig. 2).
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Figure 2: Gel Detection of Reversible ACP Labeling(a) Analysis of rhodamine-labeled crypto-ACP confirms AcpH’s ablity to remove rhodamine-pantetheine (separate lane of same gel indicated by demarcation). (b) Reversibly labeling fusion-ACPs: MBP-PaACP, GFP-ACP, and Luciferase-ACP. Apo- ACP fusions are labeled with rhodamine-CoA (mCoA) and Sfp, and subsequently unlabeled with AcpH (separate gels indicated by demarcation). (c) Acyl-pantetheine analogs were installed on ACP-15N used for NMR analysis. ACP standards for apo- and holo- allow labeling evaluation. Initial ACP-15N apo and holo mixture is readily converted to full apo- with AcpH. “One-Pot” Sfp and AcpH methodology allows conversion of one protein sample to octanoyl-, butanoyl-13C4-, and octanoyl-8-13C1-ACP-15N. Full length gels are presented in Supplementary Information (Supplementary Fig. 2).

Mentions: For evaluation of iterative labeling, we began with fluorescent ACP labeling directly in cellular lysate (Supplementary Fig. 1a) from E. coli strain DK554, which overexpresses native fatty acid ACP (AcpP) in predominantly apo- form19. Treatment of this lysate with coumarin-CoA20 and Sfp generated a blue-fluorescent band upon excitation of sodium-dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) samples at 254 nm (Supplementary Fig. 1b) that co-migrated with a coumarin-labeled ACP standard. Subsequent treatment of coumarin-labeled lysate with recombinant AcpH uniformly removed the coumarin-ppant from ACP, as demonstrated by disappearance of the blue band (Supplementary Fig. 1b). Subsequent treatment of the sample with Sfp and rhodamine-CoA21 generated a new red-fluorescent SDS-PAGE band upon excitation at 532 nm (Supplementary Fig. 1b,c); this label can also be removed (Fig. 2a, Supplementary Fig. 2) with AcpH.


Reversible labeling of native and fusion-protein motifs.

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

Gel Detection of Reversible ACP Labeling(a) Analysis of rhodamine-labeled crypto-ACP confirms AcpH’s ablity to remove rhodamine-pantetheine (separate lane of same gel indicated by demarcation). (b) Reversibly labeling fusion-ACPs: MBP-PaACP, GFP-ACP, and Luciferase-ACP. Apo- ACP fusions are labeled with rhodamine-CoA (mCoA) and Sfp, and subsequently unlabeled with AcpH (separate gels indicated by demarcation). (c) Acyl-pantetheine analogs were installed on ACP-15N used for NMR analysis. ACP standards for apo- and holo- allow labeling evaluation. Initial ACP-15N apo and holo mixture is readily converted to full apo- with AcpH. “One-Pot” Sfp and AcpH methodology allows conversion of one protein sample to octanoyl-, butanoyl-13C4-, and octanoyl-8-13C1-ACP-15N. Full length gels are presented in Supplementary Information (Supplementary Fig. 2).
© Copyright Policy
Related In: Results  -  Collection

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

Figure 2: Gel Detection of Reversible ACP Labeling(a) Analysis of rhodamine-labeled crypto-ACP confirms AcpH’s ablity to remove rhodamine-pantetheine (separate lane of same gel indicated by demarcation). (b) Reversibly labeling fusion-ACPs: MBP-PaACP, GFP-ACP, and Luciferase-ACP. Apo- ACP fusions are labeled with rhodamine-CoA (mCoA) and Sfp, and subsequently unlabeled with AcpH (separate gels indicated by demarcation). (c) Acyl-pantetheine analogs were installed on ACP-15N used for NMR analysis. ACP standards for apo- and holo- allow labeling evaluation. Initial ACP-15N apo and holo mixture is readily converted to full apo- with AcpH. “One-Pot” Sfp and AcpH methodology allows conversion of one protein sample to octanoyl-, butanoyl-13C4-, and octanoyl-8-13C1-ACP-15N. Full length gels are presented in Supplementary Information (Supplementary Fig. 2).
Mentions: For evaluation of iterative labeling, we began with fluorescent ACP labeling directly in cellular lysate (Supplementary Fig. 1a) from E. coli strain DK554, which overexpresses native fatty acid ACP (AcpP) in predominantly apo- form19. Treatment of this lysate with coumarin-CoA20 and Sfp generated a blue-fluorescent band upon excitation of sodium-dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) samples at 254 nm (Supplementary Fig. 1b) that co-migrated with a coumarin-labeled ACP standard. Subsequent treatment of coumarin-labeled lysate with recombinant AcpH uniformly removed the coumarin-ppant from ACP, as demonstrated by disappearance of the blue band (Supplementary Fig. 1b). Subsequent treatment of the sample with Sfp and rhodamine-CoA21 generated a new red-fluorescent SDS-PAGE band upon excitation at 532 nm (Supplementary Fig. 1b,c); this label can also be removed (Fig. 2a, Supplementary Fig. 2) with AcpH.

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