NMR studies of acyl chain-protein interactions

Mark Christian Oswood, Yale University.

This is an Open Access Thesis


An approach employing heteronuclear NMR is presented for monitoring the interactions between long-chain fatty acids and proteins which are involved in their synthesis and intracellular transport. Two naturally occurring fatty acids, hexadecanoic (palmitic) and cis-9-10-methylene-hexadecanoic (MHA) acids were labeled using a biosynthetic scheme with 13C acetate as a source. The biosynthetic incorporation of 13C from a two carbon source resulted in sequential pairs of 13C atoms in the product. When combined with double quantum spectroscopy, a highly efficient suppression of natural abundance background resonances results. Proton-detected two and three-dimensional experiments which correlate double quantum carbon chemical shifts with proton shifts were developed to resolve and assign chemical shifts of the selectively detected cis-9,10-methylene hexadecanoic acid (MHA) and palmitic acid in sodium dodecyl sulfate (SDS) micelles.A subset of the experiments was applied to a complex between MHA and rat adipocyte lipid binding protein (ALBP), a protein involved in intracellular fatty acid transport. With the use of double quantum spectroscopy, we were able to selectively observe and assign chemical shifts to all 13C enriched pairs of resonances in the MHA-ALBP complex. Changes in chemical shifts between micelle and protein-bound states are interpreted in terms of protein-induced conformational preferences for the bound fatty acid. Comparison with x-ray crystallographic data on similar protein-fatty acid complexes suggests that non-linear fatty acid conformations observed in the crystal structure are preserved when the complex is examined in solution.Similar double quantum experiments were successfully applied to palmitic acid covalently coupled to Escherichia coli acyl carrier protein (ACP), a protein cofactor in fatty acid synthesis. Carbon-13 chemical shifts were once again used to analyze the conformation of the acyl chain. The chain was found to have an increase in gauche character at several positions, suggesting a bent conformation in the bound state. In addition, 13C filtered cross-relaxation experiments were conducted. Cross relaxation data localized the methyl terminus of the acyl chain to a binding pocket in ACP consisting of phenylalanine-50, tyrosine-71 and isoleucine-72. These data are interpreted using the known structure of octanoyl-ACP and previous models for acyl chain binding to suggest that the thioester bond may protrude from the protein complex as the chain is lengthened in order to facilitate enzyme catalyzed reactions necessary for fatty acid synthesis.