The fatty acid synthase acyl carrier protein (AcpP) is a critical protein in the biosynthetic pathway of fatty acids by <i>E. coli.</i> AcpP transports substrates to biosynthetic enzymes by covalently attaching growing acyl chains to its 4'-phosphopantetheine (Ppant) arm, and its ability to selectively sequester acyl chains into its own hydrophobic cavity is thought to play an important role in orchestrating biosynthesis. The aim of this project is to determine whether vibrational spectroscopy can be used to determine important conformational dynamics of Ppant arm sequestration from the point of view of the inside of the protein, along the sequestration channel, as a complement to prior work that focused on vibrational probes on the arm or substrate. This will be done by inserting unnatural, 4-substituted phenylalanine amino acids with either alkyne or nitro vibrational probes at selected sites in the hydrophobic pocket of AcpP, using TAG stop codon suppression methodology to replace a native phenylalanine or tyrosine residue with the unnatural amino acid.<br/>Raman spectroscopy has a fast-enough intrinsic time scale that sequestered and non-sequestered AcpP states should be clearly distinguishable in each probe group’s spectral lineshape. The complementary reporting abilities of the nitro and alkyne unnatural amino acid vibrational probes at the same sites within hydrophobic cavity of AcpP will provide distinct Raman peaks that will likely depend differently on the changing probe environments during arm sequestration. While the nitro probe’s very strong symmetric stretching band mainly reports on the presence of water or a hydrogen-bonding environment, the alkyne probe is more hydrophobic and its strong and unique CC stretching band is suitable for contact reporting on the local electronic environment. Both of these unnatural amino acids have very strong Raman signals that should be available from relatively low-concentration samples. Because the sequestration pocket is highly hydrophobic, inserting a strongly polar nitro probe or very hydrophobic (but polarizable) alkyne probe into this pocket could interfere with overall protein structure and function. Thus, the extent to which unnatural amino acid insertion affects AcpP structure will also be investigated for this study and as a guide for future use of these specific unnatural amino acid probe groups.

Can Raman spectra of unnatural amino acids inside a protein report on functional dynamics?

ACS Spring 2022

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