Magnetic Resonance Studies of Apolipoprotein C-I Nitroxide Labeled or [¹³C]Methyl Enriched at Methionine-38

One of the three proposed lipid-binding regions of the human apolipoprotein C-I (apoC-I) is an amphipathic helix which extends from residue 33 to residue 53 and includes a single methionine at sequence position 38. The involvement of the sequence around methionine-38 in phospholipid binding has been evaluated with paramagnetic and nuclear reporter groups attached to the thiomethyl moiety. This moiety has been spin-labeled with N-(2,2,6,6-tetramethylpiperidinyl-1-oxy)bromoacetamide or ¹³C enriched with ¹³CH₃I. As determined from its EPR spectrum, the nitroxide at Met-38 of apoC-I had a rotational correlation time (τ_c) of 0.22 ns. When dimyristoylphosphatidylcholine (DMPC) was bound to the spin-labeled apoprotein, τ_c increased to 0.35 ns, indicating decreased motion for the methionyl side chain. The line width (ν_1/2) and spin-lattice relaxation time (T₁) for the thiomethyl resonance of ¹³C-enriched apoC-I in 10 mM phosphate buffer was 6.0 Hz and 320 ms, respectively. When the protein solution was made 1.6 M in Gdn·HCl, these values changed to 2.6 Hz and 970 ms, respectively. Upon addition of DMPC multilamellar liposomes to [¹³C]apoC-I in 1.6 M Gdn·HCl, the line width increased to 4.7 Hz and the T₁ decreased to 380 ms. These results strongly suggest that methionine-38 of apoC-I resides in a region of the apoprotein which undergoes significant secondary and/or tertiary structural change upon disaggregation/unfolding in Gdn·HCl and upon interaction with phospholipid.