Probing Substrate Sequestration in Carrier Proteins using Vibrational Spectroscopy Labels

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Carrier proteins are considered “lynchpin” enzymes of biosynthetic pathways. The E. coli acyl carrier protein (ACP) is composed of multiple alpha helices that form a hydrophobic, solvent-protected pocket that sometimes provides a cavernous hiding spot to protect substrates bound to the ACP's phosphopantetheine (Ppant) arm. The action of “chain sequestration” is thought to be important for driving the biosynthetic process. In this study, selected amino acid residues inside the ACP sequestration channel were targeted as possible incorporation sites for para-substituted aromatic nitrile or alkyne vibrational spectroscopic probes. Using stop codon suppression, non-canonical amino acids were incorporated into the ACP via a co-transformed aminoacyl tRNA synthetase. In addition, artificial substrates containing unique vibrational labeling groups like nitriles were covalently ligated to the ACP's Ppant arm. The vibrational frequencies of these labeling groups, measured via IR absorption or Raman scattering, report on mainly the solvent exposure of the CN group, and the lineshape reports the distribution of environments around the label on either the protein or the growing substrate. IR and Raman results that report on the sequestration of substrates of different length, as well as methodological advances that enable this spectroscopic approach, will be discussed.