Kinetics and Energetics of Assembly, Nucleation, and Growth of Aggregates and Fibrils for an Amyloidogenic Protein

Abstract

The transition states for prenucleation assembly, nucleation, and growth of aggregates and amyloid fibrils were investigated for a dimeric immunoglobulin light chain variable domain, employing pressure, temperature, and solutes as variables. Pressure-induced aggregation was nucleation-dependent and first-order in protein concentration and could be seeded. The insoluble aggregates were mixtures of amyloid fibrils and amorphous aggregates. Activation volumes, activation surface areas, and activation waters of hydration were larger for aggregate growth than for prenucleation assembly or nucleation, although activation free energies were similar for the three processes. Activation free energies for each of the transition states were dominated by the unfavorable free energy of solvation of newly exposed surfaces. Equilibrium dissociation and unfolding of the dimer showed a much larger volume change than those required to form the transition states for the three processes. Thus, the transition states for these steps are similar to the native state, and their formation requires only small structural perturbations. Finally, the presence of Congo red during amyloid fibril formation shortened lag times and caused pressure insensitivity of nucleation, suggesting that this compound or its analogs may not be effective as inhibitors of amyloidosis.

Congo Red Populates Partially Unfolded States of an Amyloidogenic Protein to Enhance Aggregation and Amyloid Fibril Formation

“…3). The second derivative IR spectra of native SMA without CR had a dominant band at 1639 cm Ϫ1 , indicating a predominance of ␤-sheet (29). During incubation at 37°C in the absence of CR, there were no changes in the secondary structure of SMA during the 14 h of the experiment.…”

“…6), which are characteristic of non-native intermolecular antiparallel ␤-sheet (6,29,33,34). Minor differences between the spectra for precipitates formed in different levels of CR may be caused by differences in morphologies of the aggregates.…”

“…Recently we showed that CR enhanced amyloid fibril formation by an amyloidogenic immunoglobulin light chain variable domain (V L ), SMA, under high pressure (29). In the present study, to gain further insight into the mechanism(s) for CR effects on amyloid fibril formation, we characterized (at atmospheric pressure) the aggregation kinetics, conformational changes, and thermal stability of SMA with a range of molar ratios of CR.…”

“…CR (Sigma C6767) solutions were prepared daily in buffer and filtered three times using a 0.45-m pore size polysulfone filter (Whatman) (29). The CR concentration was determined using an extinction coefficient of 5 (6,7).…”

“…CR birefringence under crossed-polarization microscopy and transmission electron microscopy (TEM) of precipitated proteins were performed as described previously (6,7). Precipitates formed without CR were stained with CR as described previously (29).…”