Manipulation\nand Structural Activity of AcpM in <i>Mycobacterium tuberculosis</i>

<i>Mycobacterium tuberculosis</i> (Mtb) is\na leading\ncause of death, with an escalating global occurrence of drug-resistant\ninfections that are partially attributed to cell wall mycolic acids\nderived from type II fatty acid biosynthesis (FAS-II). Here, the central\nacyl carrier protein, AcpM, contributes to the regulation of complex\nand specific protein–protein interactions (PPIs), though the\norchestration of these events remain largely unresolved due to unique\nfeatures of AcpM. Limitations include complexities in generating modified\nAcpM in a single state. Herein, we report a streamlined method to\ngenerate homogeneous samples of modified AcpM for applications in\nstructure and functional studies. We apply these to generate solvatochromic\nlabeled <i>crypto</i>-AcpM, where fluorescence response\nreports cargo sequestration and chain flipping upon interaction with\nfour FAS-II enzymes. We find an increased fluorescence in a truncated\nform, AcpM80, indicating that the 35-residue C-terminus is involved\nin modulating the chemical environment surrounding the substrate and\ncontributing to the regulation of PPIs. This study establishes an\nefficient chemo-enzymatic strategy to generate AcpM analogs for biophysical\nstudies to aid in understanding the processes driving Mtb pathogenicity\nand drug resistance.