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 (apo-C-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 reported 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 (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-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.
D053302:Apolipoprotein C-I; D001053:Apolipoproteins; D001056:Apolipoproteins C; D002942:Circular Dichroism; D003497:Cyclic N-Oxides; D004134:Dimyristoylphosphatidylcholine; D004578:Electron Spin Resonance Spectroscopy; D006801:Humans; D008081:Liposomes; D009682:Magnetic Resonance Spectroscopy; D008715:Methionine; D008745:Methylation; D008970:Molecular Weight; D010713:Phosphatidylcholines; D011487:Protein Conformation; D013113:Spin Labels
D053302:Apolipoprotein C-I; D001053:Apolipoproteins; D001056:Apolipoproteins C; D003497:Cyclic N-Oxides; D008081:Liposomes; D010713:Phosphatidylcholines; D013113:Spin Labels; C028369:tempo 4-bromoacetamide; D008715:Methionine; D004134:Dimyristoylphosphatidylcholine