Fluorescence correlation spectroscopy shows that monomeric polyglutamine molecules form collapsed structures in aqueous solutions

Abstract

We have used fluorescence correlation spectroscopy measurements to quantify the hydrodynamic sizes of monomeric polyglutamine as a function of chain length (N) by measuring the scaling of translational diffusion times (tau(D)) for the peptide series (Gly)-(Gln)(N)-Cys-Lys(2) in aqueous solution. We find that tau(D) scales with N as tau(o)N(nu) and therefore ln(tau(D)) = ln(tau(o)) + nuln(N). The values for nu and ln(tau(o)) are 0.32 +/- 0.02 and 3.04 +/- 0.08, respectively. Based on these observations, we conclude that water is a polymeric poor solvent for polyglutamine. Previous studies have shown that monomeric polyglutamine is intrinsically disordered. These observations combined with our fluorescence correlation spectroscopy data suggest that the ensemble for monomeric polyglutamine is made up of a heterogeneous collection of collapsed structures. This result is striking because the preference for collapsed structures arises despite the absence of residues deemed to be hydrophobic in the sequence constructs studied. Working under the assumption that the driving forces for collapse are similar to those for aggregation, we discuss the implications of our results for the thermodynamics and kinetics of polyglutamine aggregation, a process that has been implicated in the molecular mechanism of Huntington's disease.

Fig. 1.

Variation of 〈τ_D〉 with 〈N〉, the average number of glutamine residues in the peptide series Gly-(Gln)_〈N〉-Cys*-(Lys)₂. Horizontal error bars are asymmetric because they are not true error bars. Instead, they are meant to convey the range of chain lengths within each peptide sample. The ordinate labels shown in italics are estimates for the hydrodynamic radius (〈R_h〉) in Å, which were calculated from measured values for 〈τ_D〉 by using the prescription described in Methods.

Fig. 2.

Plot of ln〈τ_D〉 as a function of ln(〈N〉). The solid line is the line of best fit to FCS data shown as open diamonds, and the dotted lines represent the 95% confidence intervals. Error bars are standard errors in our estimate for the mean value of ln〈τ_D〉, which is obtained from Monte Carlo bootstrap analysis (see Methods).

Fig. 3.

Schematic of the free energy surface G(R_g, Z_β) for a single polyglutamine chain. The radius of gyration (R_g) and overall β-content (Z_β) are the two reaction coordinates. The horizontal dashed line is meant to denote the distinction between collapsed and extended structures. The third reaction coordinate, n, denotes monomer addition. If a single chain acts as the critical nucleus, then monomer addition is a downhill process and conformational changes within the monomer are either rate-limiting or thermodynamically unfavorable. Accordingly, the dominant free energy minima are shown as being compact with very few internal β-sheet contacts. The blue stars denote free energy maxima, which are putative nuclei for model 1. The red star shows the critical nucleus for model 2. This is a free energy maximum on account of being partially swollen and is different from other partially swollen conformations because of the extent of internal β-sheet formation. Finally, the compact β-sheet, which is the model proposed by Chen et al. (16) is shown as a green star. In the color bar, as intramolecular free energies increase, the colors change from blue to red. The dashed arrows from the different putative nuclei to the fibril are meant to depict the proposal that if the critical nucleus is a single chain, monomer addition is downhill in free energy.