Mirco Sorci1, Arpan Nayak1, Chuang-Chung Lee2, Gregory J. McRae2, Robert Grassucci3, Ingrid Hahn3, Joachim Frank3, and Georges Belfort1. (1) Rensselaer Polytechnic Institute, 110 8th st, Troy, NY 12180-3590, (2) MIT, Cambridge, MA 02141, (3) Wadsworth Center and Howard Hughes Medical Institute
Amyloid fibrillation is the process of conversion of native soluble proteins into insoluble fibrils comprising of cross β-sheets and has received wide attention due to its substantial physiological relevance and to the complexity of the underlying physical and chemical reactions. In our group, we have performed fibrillation experiments using recombinant insulin; a safe protein that follows the typical sigmoid curve common for other amyloids. It comprises an initial lag phase, a subsequent growth step, and saturation to a plateau. The growth step has been characterized previously and in our group using AFM imaging analysis. In this work, attention is focused on the lag-phase and concomitant oligomer formation that some consider to be the toxic agents during amyloidosis. Several methods are used to analyze the oligomers including small angle neutron scattering, sucrose gradient centrifugation, cryo-electron microscopy and “re-heating experiments”. This last set of experiments comprised three steps: (i) heating the samples at 65°C and pH 1.6 for a given period, (ii) removing the samples from the heater and keeping them at 25°C for a “waiting period”, and (iii) “re-heating” the samples at 65°C for another period. The fibrillation process was followed with absorbance measurements. The “waiting period” was introduced in order to evaluate the reversibility of the oligomerization process during the lag phase. The results confirmed that the aggregates formed during the first “heating” step were very stable after 24 hours. However, as the “waiting period” was increased, the aggregates appeared to lose memory of their previous treatment, resulting in longer and longer lag-times, eventually reverting back to the reference curve. After a month of waiting, a relative increase of 57% was noted and using a fitting equation we predicted a “waiting period” of 55 days to revert back to the characteristic lag-time of the reference curve. “The re-heating experiments” have provided a simple tool to prove the stability and reversibility of the oligomerization process prior to fibril formation.