Magnetic resonance studies of apolipoprotein C-I nitroxide-labeled or [13C]methyl enriched at methionine-38

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Abstract
One of the 3 proposed lipid-binding regions of the human apolipoprotein C-I (apoC-P) 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 was evaluated with paramagnetic and nuclear reporter groups attached to the thiomethyl moiety. This moiety was 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 (.tau.c) of 0.22 ns. When dimyristoylphosphatidylcholine (DMPC) was bound to the spin-labeled apoprotein, .tau.c increased to 0.35 ns, indicating decreased motion for the methionyl side chain. The line width (.nu.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[guanidine].cntdot.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.cntdot.HCl, the line width increased to 4.7 Hz and the T1 decreased to 380 ms. 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.cntdot.HCl and upon interaction with phospholipid.