Polyglutamine aggregation nucleation: Thermodynamics of a highly unfavorable protein folding reaction

Abstract

Polyglutamine (polyGln) aggregation is implicated in the disease progression of Huntington's disease and other expanded CAG repeat diseases. PolyGln aggregation in vitro follows a simple nucleated growth polymerization pathway without apparent complications such as populated intermediates, alternative assembly pathways, or secondary nucleation phenomena. Previous analysis of the aggregation of simple polyGln peptides revealed that the critical nucleus (the number of monomeric units involved in the formation of an energetically unfavorable aggregation nucleus) is equal to one, suggesting that polyGln nucleation can be viewed as an unfavorable protein folding reaction. We provide here a method for experimentally determining the number of elongation growth sites per unit weight for any polyGln aggregate preparation, a key parameter required for completion of the nucleation kinetics analysis and determination of the thermodynamics of nucleation. We find that, for the polyGln peptide Q 47, the secondorder rate constant for fibril elongation is 11,400 liters͞mol per s, whereas K n*, the equilibrium constant for nucleation of aggregation, is remarkably small, equal to 2.6 ؋ 10 ؊9 . The latter value corresponds to a free energy of nucleus formation of ؉12.2 kcal͞mol, a value consistent with a highly unfavorable folding reaction. The methods introduced here should allow further analysis of the energetics of polyGln nucleus formation and accurate comparisons of the seeding capabilities of different fibril preparations, a task of increasing importance in the amyloid field.amyloid ͉ Huntington's disease ͉ nucleated growth polymerization ͉ nucleation free energy

Dynamic Imaging by Fluorescence Correlation Spectroscopy Identifies Diverse Populations of Polyglutamine Oligomers Formed in Vivo

“…These studies show that oligomers and immobile aggregates both appear in a time dependent manner, a result that is consistent with a nucleation-dependent reaction observed in vitro for many aggregation-prone proteins (14,27,66,67), and the appearance of regions comprised of oligomers and monomers that appear diffuse in vivo (68 -70). While some in vitro studies have suggested a predominant oligomer species (30,71,72), others indicate the appearance of a heterogeneous distribution of oligomers (63,73,74).…”

A polymer physics perspective on driving forces and mechanisms for protein aggregation

“…Experiments have also shown that the tendency to form intermolecular aggregates increases with polyglutamine length. Wetzel and coworkers have carried out a series of experiments based on a variety of spectroscopic and other probes to quantify the overall rate of aggregation as a function of chain length and concentration [45,54,[65][66][67]. Their conclusions are consistent, at least qualitatively, with the expectations from polymer theory as discussed above and demonstrated in Figure 3.…”

“…Despite the impressive progress made by Wetzel [45,54,[65][66][67] and others [68][69][70][71][72] in the area of polyglutamine aggregation, there are significant gaps in our knowledge because not much is known about intermolecular associations and intramolecular conformational fluctuations that occur at very low protein concentrations (nM -μM). This knowledge is of utmost importance to understand the phenomenon of polyglutamine aggregation in vivo.…”

Kinetic mechanism of p53 oncogenic mutant aggregation and its inhibition

“…Such curves can be fitted to empirical equations. Following the procedure of Wetzel and coworkers (25,26), we used a simple model-free method and plotted the initial rates of scattering against t 2 , where t is time [a generally useful plot (27)]. The rate of addition of monomer to growing oligomers varies as t 2 and the slope of the plot is V 0 .…”