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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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HSQC spectra of recycled ACP-15N in various acyl states overlayed with apo-ACP-15NAll spectra were collected on the same protein sample. (a) 15N-1H -HSQC of the originally prepared apo-ACP-15N (black) is overlayed with the HSQC of octanoyl-ACP-15N (blue). (b) HSQC of regenerated apo-ACP-15N (red) overlayed with the original apo-ACP-15N preparation (black). (c) HSQC of butanoyl-13C4-ACP-15N (orange) is overlayed with the original apo-ACP-15N (black). (d) HSQC of octanoyl-8-13C1-ACP-15N (lavender) is overlayed with the original apo-ACP-15N (black). Full spectra are available in Supplementary Information (Supplemtary Figs. 9-14).
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Figure 3: HSQC spectra of recycled ACP-15N in various acyl states overlayed with apo-ACP-15NAll spectra were collected on the same protein sample. (a) 15N-1H -HSQC of the originally prepared apo-ACP-15N (black) is overlayed with the HSQC of octanoyl-ACP-15N (blue). (b) HSQC of regenerated apo-ACP-15N (red) overlayed with the original apo-ACP-15N preparation (black). (c) HSQC of butanoyl-13C4-ACP-15N (orange) is overlayed with the original apo-ACP-15N (black). (d) HSQC of octanoyl-8-13C1-ACP-15N (lavender) is overlayed with the original apo-ACP-15N (black). Full spectra are available in Supplementary Information (Supplemtary Figs. 9-14).

Mentions: We evaluated ACP-15N at each labeling conformation via gel (Fig. 2c and Supplementary Figs. 7 and 8) and NMR analysis (Supplementary Figs. 9-14). We obtained an initial apo and holo mixture following E. coli expression, requiring full conversion to the apo- form (Fig. 2c) using AcpH. Subsequent conversion to octanoyl-ACP-15N (Fig. 2c) utilized the chemo-enzymatic synthesis of octanoyl-CoA14 with Sfp labeling. After NMR evaluation, we converted the ACP-15N back to the apo- form with AcpH for subsequent relabeling. We acquired 15N-1H heteronuclear single quantum coherence (HSQC) spectra of all three ACP-15N species (apo-, octanoyl-, and regenerated apo-). Comparing apo-ACP-15N to the octanoyl-ACP-15N (Fig. 3a), we observed chemical shift perturbations characteristic of acyl chain sequestration in the hydrophobic binding pocket26. Conversion from this acylated form back to the apo- form by AcpH provided uniformly unlabeled apo-ACP-15N, as confirmed by an HSQC spectrum of the regenerated protein that identically matched that of the original (Fig. 3b). This validated the feasibility of reversible ACP labeling, as it demonstrated that the regenerated apo-ACP-15N is properly folded and ready for subsequent modification.


Reversible labeling of native and fusion-protein motifs.

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

HSQC spectra of recycled ACP-15N in various acyl states overlayed with apo-ACP-15NAll spectra were collected on the same protein sample. (a) 15N-1H -HSQC of the originally prepared apo-ACP-15N (black) is overlayed with the HSQC of octanoyl-ACP-15N (blue). (b) HSQC of regenerated apo-ACP-15N (red) overlayed with the original apo-ACP-15N preparation (black). (c) HSQC of butanoyl-13C4-ACP-15N (orange) is overlayed with the original apo-ACP-15N (black). (d) HSQC of octanoyl-8-13C1-ACP-15N (lavender) is overlayed with the original apo-ACP-15N (black). Full spectra are available in Supplementary Information (Supplemtary Figs. 9-14).
© Copyright Policy
Related In: Results  -  Collection

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

Figure 3: HSQC spectra of recycled ACP-15N in various acyl states overlayed with apo-ACP-15NAll spectra were collected on the same protein sample. (a) 15N-1H -HSQC of the originally prepared apo-ACP-15N (black) is overlayed with the HSQC of octanoyl-ACP-15N (blue). (b) HSQC of regenerated apo-ACP-15N (red) overlayed with the original apo-ACP-15N preparation (black). (c) HSQC of butanoyl-13C4-ACP-15N (orange) is overlayed with the original apo-ACP-15N (black). (d) HSQC of octanoyl-8-13C1-ACP-15N (lavender) is overlayed with the original apo-ACP-15N (black). Full spectra are available in Supplementary Information (Supplemtary Figs. 9-14).
Mentions: We evaluated ACP-15N at each labeling conformation via gel (Fig. 2c and Supplementary Figs. 7 and 8) and NMR analysis (Supplementary Figs. 9-14). We obtained an initial apo and holo mixture following E. coli expression, requiring full conversion to the apo- form (Fig. 2c) using AcpH. Subsequent conversion to octanoyl-ACP-15N (Fig. 2c) utilized the chemo-enzymatic synthesis of octanoyl-CoA14 with Sfp labeling. After NMR evaluation, we converted the ACP-15N back to the apo- form with AcpH for subsequent relabeling. We acquired 15N-1H heteronuclear single quantum coherence (HSQC) spectra of all three ACP-15N species (apo-, octanoyl-, and regenerated apo-). Comparing apo-ACP-15N to the octanoyl-ACP-15N (Fig. 3a), we observed chemical shift perturbations characteristic of acyl chain sequestration in the hydrophobic binding pocket26. Conversion from this acylated form back to the apo- form by AcpH provided uniformly unlabeled apo-ACP-15N, as confirmed by an HSQC spectrum of the regenerated protein that identically matched that of the original (Fig. 3b). This validated the feasibility of reversible ACP labeling, as it demonstrated that the regenerated apo-ACP-15N is properly folded and ready for subsequent modification.

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