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).