Effects of the Iowa and Milano Mutations on Apolipoprotein A-I Structure and Dynamics Determined by Hydrogen Exchange and Mass Spectrometry

摘要

The Iowa point mutation in apolipoprotein A-I (G26R; apoA-IIowa) leads to a systemic amyloidosis condition and the Milano mutation (R173C; apoA-IMil) is associated with hypoalphalipoproteinemia, a reduced plasma level of high density lipoprotein. To probe the structural effects that lead to these outcomes, we used amide hydrogen-deuterium exchange coupled with a fragment separation/mass spectrometry analysis (HX MS). The Iowa mutation inserts an arginine residue into the non-polar face of an α-helix that spans residues 7–44 and causes changes in structure and structural dynamics. This helix unfolds and other helices in the N-terminal helix bundle domain are destabilized. The segment encompassing residues 116–158, largely unstructured in wild type apoA-I, becomes helical. The helix spanning residues 81 to 115 is destabilized by 2 kcal/mol, increasing the small fraction of time it is transiently unfolded to 1% or more, which allows proteolysis at residue 83 in vivo over time, releasing an amyloid-forming peptide. The Milano mutation situated on the polar face of the helix spanning residues 147–178 destabilizes the helix bundle domain only moderately, but enough to allow cysteine-mediated dimerization which leads to the altered functionality of this variant. These results show how the HX MS approach can provide a powerful means for monitoring, in a non-perturbing way and at close to amino acid resolution, the structural, dynamic, and energetic consequences of biologically interesting point mutations.