Mycobacterium tuberculosis (Mtb) is a leading cause of death, with an escalating global occurrence of drug-resistant infections that are partially attributed to cell wall mycolic acids derived from type II fatty acid biosynthesis (FAS-II). Here, the central acyl carrier protein, AcpM, contributes to the regulation of complex and specific protein-protein interactions (PPIs), though the orchestration of these events remain largely unresolved due to unique features of AcpM. Limitations include complexities in generating modified AcpM in a single state. Herein, we report a streamlined method to generate homogeneous samples of modified AcpM for applications in structure and functional studies. We apply these to generate solvatochromic labeled crypto-AcpM, where fluorescence response reports cargo sequestration and chain flipping upon interaction with four FAS-II enzymes. We find an increased fluorescence in a truncated form, AcpM80, indicating that the 35-residue C-terminus is involved in modulating the chemical environment surrounding the substrate and contributing to the regulation of PPIs. This study establishes an efficient chemo-enzymatic strategy to generate AcpM analogs for biophysical studies to aid in understanding the processes driving Mtb pathogenicity and drug resistance.