Design, synthesis, and evaluation of an isotopic labeling strategy for studying fatty acid-protein binding by NMR.
Here we describe the synthesis and evaluation of a new isotopic labeling strategy for fatty acids to be used as probes for studying ligand binding by NMR. We synthesized palmitic acid with carbons C-3 through C-16 perdeuterated, C-1 and C-2 with 13C atoms and hydrogens at C-2. Our strategy began with commercially available perdeuterated myristic acid and built up to palmitic acid using a Horner-Wadsworth-Emmons reaction. To evaluate the power of this isotopic enrichment strategy, we evaluated ligand binding to a prototypical member of the intracellular lipid binding protein family, FABP2. This small 15 kDa protein is well known to bind fatty acids with high affinity, and we used this system to illustrate the spectral filtering abilities of our described labeling strategy. Herein we show how having two vicinal 13C-enriched carbons, two hydrogens at the alpha-position, and a perdeuterated aliphatic tail allows the efficient use of multidimensional NMR experiments to effectively filter all background resonances from the protein and facilitate the study of ligand binding.
Han
Yong
Y
Department of Chemistry, Case Western Reserve University, 10900 Euclid Avenue, Cleveland, Ohio 44106, USA.
Alexander
Timothy E
TE
Tochtrop
Gregory P
GP
D001665:Binding Sites; D002247:Carbon Isotopes; D050556:Fatty Acid-Binding Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000187:drug effects; D006801:Humans; D007553:Isotope Labeling / Q000379:methods*; D008024:Ligands; D009682:Magnetic Resonance Spectroscopy / Q000379:methods* / Q000592:standards; D015394:Molecular Structure; D019814:Myristic Acid / Q000737:chemistry; D019308:Palmitic Acid / Q000138:chemical synthesis / Q000737:chemistry* / Q000494:pharmacology; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000187:drug effects; D012015:Reference Standards; D013237:Stereoisomerism
D002247:Carbon Isotopes; C493404:FABP2 protein, human; D050556:Fatty Acid-Binding Proteins; D008024:Ligands; D011994:Recombinant Proteins; D019814:Myristic Acid; D019308:Palmitic Acid