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 second-order rate constant for fibril elongation is 11,400 liters/mol per s, whereas K(n*)), the equilibrium constant for nucleation of aggregation, is remarkably small, equal to 2.6 x 10(-9). The latter value corresponds to a free energy of nucleus formation of +12.2 kcal/mol, 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.

Fig. 1.

Nucleated growth polymerization with monomer addition. (a) Previously described mechanism in which nucleation is an unfavorable folding event within monomeric polyGln; adapted from ref. . (b) Addition of tagged monomer to the growing end of a polyGln aggregate is unstable and prone to dissociate unless followed by polymer growth through subsequent rounds of monomer addition.

Fig. 2.

Time-dependent binding of biotinylated peptides to aggregates. (a) Binding of biotin-PEG-Q₂₉ to Q₄₇ aggregates grown in PBS at 37°C (squares). (b) Binding of biotin-PEG-Q₂₉ to Q₄₇ aggregates grown in Tris·HCl at 37°C (diamonds). In a and b,Q₄₇ aggregates were sonicated (empty symbols) or not (filled symbols). Each time point contained 500 μl of either 190 ng/ml (squares) or 260 ng/ml (diamonds) aggregate and was developed by incubation 15 min at 25°C with 30 μM unlabeled Q_30. Controls are Q₄₇ aggregates with (○) or without (•) sonication, with no added unlabeled Q₃₀ during workup (see Materials and Methods). (c) Binding of heterogeneous biotin-labeled peptides to Q₄₇ (PBS, 37°C) aggregates: 0.5 μM biotin-PEG-PGQ₉-P^2,3, with (▵) or without (○)30 μM unlabeled Q₃₀ added during work-up; 0.1 μM biotin-Aβ(1-40), with 5 μM unlabeled Aβ(1-40) added during work-up (▴). In c, all time points consisted of 500 μl of 228 ng/ml Q₄₇ aggregates.

Fig. 3.

Time and concentration dependence of biotinyl-Q₂₉ binding to Q₄₇ aggregates. (a and b) Biotin-PEG-Q₂₉ (0.5 μM) was incubated with 240 ng/ml Q₄₇ aggregates at 25°C for 20 min, then 30 μM unlabeled Q₃₀ monomer was added and the reaction shifted to 37°C. □, fmol biotin-PEG-Q₂₉ bound, as stabilized by added unlabeled Q₃₀; ○, fmol biotin-PEG-Q₂₉ bound, in control lacking unlabeled Q₃₀; ♦, percentage of unlabeled Q₃₀ remaining monomeric in a centrifugation supernatant, as determined by analytical HPLC. (c) Amount of biotinyl-PEG-Q₂₉ bound in 30 min at 25°C.

Fig. 4.

Correlation of experimentally determined growing ends to pseudofirst-order rate constants for elongation. (a) Determination of k^*, the pseudofirst-order rate constant for elongation of Q₄₇ monomers by Q₄₇ aggregates (grown in PBS, 37°C, and nonsonicated), with unpolymerized monomer determined by the HPLC sedimentation assay. (b) For six different aggregates (see Table 1), correlation of growing end concentration of an aggregate suspension and the pseudofirst-order elongation rate constant for that aggregate suspension mixed with monomeric polyGln (aggregates made in PBS, ♦; aggregates made in Tris·HCl, ▪).

Fig. 5.

Estimation of growing ends by using a microtiter plate elongation assay. Time-dependent addition of biotin-PEG-Q₂₉ to various Q₄₇ aggregates adhered to plastic microtiter plate wells: PBS, 37°C aggregates (squares); Tris·HCl, 37°C aggregates (diamonds); with (empty symbols) and without (filled symbols) sonication. (a) Overall time course. (b) Extrapolation of slow, second kinetic phase back to zero time to obtain the approximate amplitude of the fast phase.

Fig. 6.

Analysis of nucleation kinetics. (a) Representative time² plots of the early portion of Q₄₇ (PBS, 37°C) at different concentrations (▴, 36 μM, left y axis; •, 105 μM, right y axis). (b) Log-log plot of the slopes of the time² plots vs. Q₄₇ concentration. The slope of the log-log plot is 2.87 (R² = 0.977).