Ultraviolet Photodissociation Mass Spectrometry Captures the Acyl Chain Length-Dependent Conformation Dynamics of Acyl Carrier Protein.

Capturing the acyl chain dependent conformation dynamics of acyl carrier protein (ACP) is critical for understanding the molecular mechanism of acyl chain stabilization and elongation, providing structural insights for ACP evolution. Herein, we utilize native mass spectrometry (nMS) and 193 nm ultraviolet photodissociation (UVPD) to systematically interrogate the structural details in activation and interactions of ACP with C4-C18 acyl chains. The unstable acyl-ACP intermediates can be isolated and subjected to high-sensitivity UVPD analysis individually without matrix interference. We find that the acyl chains mainly insert into ACP subpocket I until the chain length surpasses the cavity's maximum capacity by 10 carbons. Then, the hydrophobic part of long acyl chains (>C10) bends into subpocket II. Notably, Phe50 and Ile62 play a critical role in regulating the size of the hydrophobic pocket, while Loop I and Thr64-Gln66 are essential for stabilizing long-chain acyl-ACPs. Our findings pave the way for ACP rational evolution to promote the biosynthesis of target fatty acids.