Resin-based investigation of acyl carrier protein interaction networks in Escherichia coli

Abstract

Protein-protein interactions play an integral role in metabolic regulation. Elucidation of these networks is complicated by the changing identity of the proteins themselves. Here we demonstrate a resin-based technique that leverages the unique tools for acyl carrier protein (ACP) modification with non-hydrolyzable linkages. ACPs from Escherichia coli and Shewanella oneidensis MR-1 are bound to Affigel-15 with varying acyl groups attached and introduced to proteomic samples. Isolation of these binding partners is followed by MudPIT analysis to identify each interactome with the variable of ACP-tethered substrates. These techniques allow for investigation of protein interaction networks with the changing identity of a given protein target.

Figure 1

Probes used. (a) Coenzyme A is linked to a rhodamine analog through a maleamide linkage and used as a fluorescent ACP probe. (b) Crypto-pantetheine probes including an octanoyl probe 1, lauroyl probe 2 and trans-chloroacryloyl probe 3. Each probe 1–3 is ‘non-hydrolyzable’ in media due to the presence of a stronger amide linkage, as compared to the thioester bond commonly occurring in natural acyl-CoAs.

Figure 2

Preparation of non-hydrolyzable acyl-ACP analogs. (a) Probes 1 and 2 were appended enzymatically to apo-ACP and purified using an orthogonal purification strategy. After derivitization, the mixture was batch-bound to Ni-NTA resin. Low concentration imidazole washes removed CoA biosynthetic enzymes with maltose binding protein fusion tags and untagged Sfp. Pure, His₆ tagged ACP was then eluted by application of 300 mM imidazole. Side chain R is as in Figure 1. (b) Attachment by addition (+) of 1, 2 or Coenzyme A to ACP was visualized by Urea–PAGE analysis. The (−) sign denotes that the compound was left out of the reaction mixture.

Figure 3

Visualization of resin-bound ACP using fluorescence microscopy. Images denote white light (left) and fluorescence (right) from the same sample. Images (a) and (b) show crypto-ACP beads incubated with MBP-CoaA, MBP-CoaD, MBP-CoaE, native Sfp and KASII-GFP. Crosslinking probe chloroacrylate pantetheine 3 is also introduced in (b) resulting in a covalent adduct between ACP and KASII-GFP and a green fluorescence observed by excitation at 475 ± 30 nm. Compound 3 is omitted in negative control (a). Images (c) and (d) show crypto-ACP beads incubated with Rhodamine-mCoA. Sfp is also introduced in (d) resulting in fluorescent labeling and a red fluorescence observed by excitation at 570 ± 35 nm. Sfp is omitted in negative control (c). The white bar denotes 300 μm.

Figure 4

Synthetic route to lauroyl pantetheine (2) (a) Lauroyl chloride (1.1 equiv), pyridine (1 equiv), DCM, 2 h. (b) 1 M HCl/THF, 1 h.