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 ( D) for the peptide series (Gly)-(Gln)N-Cys-Lys2 in aqueous solution. We find that D scales with N as oN and therefore ln( D) ‫؍‬ ln( o) ؉ ln(N). The values for and ln( 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.chain collapse ͉ poor solvent T he accumulation of ordered intracellular and extracellular protein aggregates are visible molecular characteristics of a variety of neurodegenerative and systemic diseases (1-6). Nine neurodegenerative diseases, including Huntington's disease, are associated with the aggregation of proteins that contain genetic expansions of polyglutamine tracts above a normal threshold length of 35 glutamine residues (7-10). Ages of onset of disease show nonlinear, inverse correlation with the length of polyglutamine expansions (11). Different hypotheses have been put forth to explain both the toxicity associated with polyglutamine expansions and what it is about the expansion above a normal length range that confers toxicity. A majority of proposed mechanisms center on the aggressive, length-dependent, ability of polyglutamine to form ordered intermolecular aggregates (12).CD and NMR data indicate that monomeric polyglutamine sequences prefer the random coil state under physiological conditions (12-15). As chain length increases, there is no obvious change in the ensemble averaged solution ''structure'' of polyglutamine peptides (14) although data from different in vitro experiments indicate that rates of aggregation increase with polyglutamine length (14, 16). Mechanistically, polyglutamine aggregation is a nucleation-dependent process (16), and analysis of kinetic data using a thermodynamic nucleus model (17) suggests that an energetically unfavorable conformation of the monomer, i.e., a single polyglutamine chain, acts as the critical nucleus for aggregation (16,18).It is important to recognize that one can invoke a range of mechanisms to explain kinetic data for polypeptide aggregation (5, 16, 17) and the dominant mechanis...

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