Measuring the Energy Barrier of the Structural Change That Initiates Amyloid Formation.

Obtaining kinetic and thermodynamic information for protein amyloid formation can yield new insight into the mechanistic details of this biomedically important process. The kinetics of the structural change that initiates the amyloid pathway, however, has been challenging to access for any amyloid protein system. Here, using the protein ß-2-microglobulin (ß2m) as a model, we measure the kinetics and energy barrier associated with an initial amyloidogenic structural change. Using covalent labeling and mass spectrometry, we measure the decrease in solvent accessibility of one of ß2m's Trp residues, which is buried during the initial structural change, as a way to probe the kinetics of this structural change at different temperatures and under different amyloid forming conditions. Our results provide the first-ever measure of the activation barrier for a structural change that initiates the amyloid formation pathway. The results also yield new mechanistic insight into ß2m's amyloidogenic structural change, especially the role of Pro32 isomerization in this reaction.

Increased ß-Sheet Dynamics and D-E Loop Repositioning Are Necessary for Cu(II)-Induced Amyloid Formation by ß-2-Microglobulin.

ß-2-Microglobulin (ß2m) forms amyloid fibrils in the joints of patients undergoing dialysis treatment as a result of kidney failure. One of the ways in which ß2m can be induced to form amyloid fibrils in vitro is via incubation with stoichiometric amounts of Cu(II). To better understand the structural changes caused by Cu(II) binding that allow ß2m to form amyloid fibrils, we compared the effect of Ni(II) and Zn(II) binding, which are two similarly sized divalent metal ions that do not induce ß2m amyloid formation. Using hydrogen/deuterium exchange mass spectrometry (HDX/MS) and covalent labeling MS, we find that Ni(II) has little effect on ß2m structure, despite binding in the same region of the protein as Cu(II). This observation indicates that subtle differences in the organization of residues around Cu(II) cause distant changes that are necessary for oligomerization and eventual amyloid formation. One key difference that we find is that only Cu(II), not Ni(II) or Zn(II), is able to cause the cis-trans isomerization of Pro32 that is an important conformational switch that initiates ß2m amyloid formation. By comparing HDX/MS data from the three metal-ß2m complexes, we also discover that increased dynamics in the ß-sheet formed by the A, B, D, and E ß strands of the protein and repositioning of residues in the D-E loop are necessary aspects of ß2m forming an amyloid-competent dimer. Altogether, our results reveal new structural insights into the unique effect of Cu(II) in the metal-induced amyloid formation of ß2m.