Probing Substrate Sequestration in Carrier Proteins using Vibrational Spectroscopy Labels and Molecular Dynamic Simulations

Abstract

Carrier proteins are considered “lynchpin” enzymes of biosynthetic pathways. The E. coli acyl carrier protein (ACP) has multiple alpha helices that form a hydrophobic, solvent-protected pocket and can provide a hiding spot to protect growing substrates bound to the ACP's phosphopantetheine (Ppant) arm. The action of “chain sequestration” is thought to be important for driving the acyl chain biosynthetic process. Site specific vibrational spectroscopy techniques are being used to investigate Ppant arm dynamics via nitrile probes on unnatural amino acids incorporated into the sequestration cavity. Molecular dynamics simulations in aim to characterize the Ppant arm's shifting conformational distribution and substrate-loading selectivity by analyzing geometric quantities and the solvent exposed surface area (SASA) of specific pieces of the arm. Ultimately, incorporating a nitrile probe group into the simulation will provide a direct connection to experimental data through the CN infrared spectroscopic lineshape. A thorough understanding of Ppant arm dynamics will inform further characterization of experimental data from carrier proteins and efforts to manipulate ACP's interactions with partner enzymes.