Intrinsically disordered proteins (IDPs) adopt heterogeneous ensembles of conformations under physiological conditions. Understanding the relationship between amino acid sequence and conformational ensembles of IDPs can help clarify the role of disorder in physiological function. Recent studies revealed that polar IDPs favor collapsed ensembles in water despite the absence of hydrophobic groups-a result that holds for polypeptide backbones as well. By studying highly charged polypeptides, a different archetype of IDPs, we assess how charge content modulates the intrinsic preference of polypeptide backbones for collapsed structures. We characterized conformational ensembles for a set of protamines in aqueous milieus using molecular simulations and fluorescence measurements. Protamines are arginine-rich IDPs involved in the condensation of chromatin during spermatogenesis. Simulations based on the ABSINTH implicit solvation model predict the existence of a globule-to-coil transition, with net charge per residue serving as the discriminating order parameter. The transition is supported by quantitative agreement between simulation and experiment. Local conformational preferences partially explain the observed trends of polymeric properties. Our results lead to the proposal of a schematic protein phase diagram that should enable prediction of polymeric attributes for IDP conformational ensembles using easily calculated physicochemical properties of amino acid sequences. Although sequence composition allows the prediction of polymeric properties, interresidue contact preferences of protamines with similar polymeric attributes suggest that certain details of conformational ensembles depend on the sequence. This provides a plausible mechanism for specificity in the functions of IDPs.Monte Carlo | polyampholyte | polyelectrolyte I ntrinsically disordered proteins (IDPs) are a class of proteins that fail to fold autonomously in aqueous solutions to welldefined three-dimensional structures (1, 2). This "intrinsic disorder" has been implicated in a range of regulatory functions that require IDPs to interact with other macromolecular ligands (3-11). Many of these interactions promote disorder-to-order transitions within IDPs (3, 9), and different mechanistic models (3, 12, 13) have been proposed for coupled folding and binding. To develop a better understanding of how disorder is used in function (2), we have pursued quantitative, polymer-physics-based descriptions (14-16) for conformational ensembles of .Low hydrophobicity is a defining characteristic of IDP sequences (21,22). This suggests that IDPs cannot collapse to form compact, globular conformations in aqueous solutions (21). However, spectroscopic (23-27) and computational investigations (17, 28) have shown that polar tracts form heterogeneous ensembles of collapsed structures in aqueous solutions. These sequences are rich in uncharged, polar amino acids and are devoid of canonical hydrophobic residues. Collapse of polar tracts has been observed for polyglutamine (17...

One of the neuropathological hallmarks of Alzheimer's disease (AD) is the amyloid plaque, primarily composed of aggregated amyloidbeta (A␤) peptide. In vitro, A␤1-42, the major alloform of A␤ found in plaques, self-assembles into fibrils at micromolar concentrations and acidic pH. Such conditions do not exist in the extracellular fluid of the brain where the pH is neutral and A␤ concentrations are in the nanomolar range. Here, we show that extracellular soluble A␤ (sA␤) at concentrations as low as 1 nM was taken up by murine cortical neurons and neuroblastoma (SHSY5Y) cells but not by human embryonic kidney (HEK293) cells. Following uptake, A␤ accumulated in Lysotracker-positive acidic vesicles (likely late endosomes or lysosomes) where effective concentrations (>2.5 M) were greater than two orders of magnitude higher than that in the extracellular fluid (25 nM), as quantified by fluorescence intensity using laser scanning confocal microscopy. Furthermore, SHSY5Y cells incubated with 1 M A␤1-42 for several days demonstrated a time-dependent increase in intracellular high molecular weight (HMW) (>200 kDa) aggregates, which were absent in cells grown in the presence of A␤1-40. Homogenates from these A␤1-42-loaded cells were capable of seeding amyloid fibril growth. These results demonstrate that A␤ can be taken up by certain cells at low physiologically relevant concentrations of extracellular A␤, and then concentrated into endosomes/lysosomes. At high concentrations, vesicular A␤ aggregates to form HMW species which are capable of seeding amyloid fibril growth. We speculate that extrusion of these aggregates may seed extracellular amyloid plaque formation during AD pathogenesis.amyloid fibrils ͉ late endosomes ͉ lysosomes ͉ plaques A lzheimer's disease (AD), the most common form of dementia in Western countries, involves progressive accumulation of amyloid deposits, neuronal loss, cognitive decline, and eventual death. Senile plaques, a key pathological feature of this disease, are composed primarily of the amyloid-beta (A␤) peptide, and are found throughout the brain (1). A␤ (ranging in length from 39-42 amino acids) is derived from the proteolytic cleavage of an endogenous transmembrane protein known as the amyloid precursor protein (APP). The most common A␤ peptide found in senile plaques is the 42-residue peptide (A␤ 1-42 ) (2), which also shows the strongest propensity for spontaneous aggregation in solution (3). It is widely believed that the aggregation and accumulation of this peptide is involved in disease pathogenesis.A␤ is produced primarily by neurons and secreted into the brain extracellular space where it is normally found in a soluble state (4). A variety of physiological processes, including those associated with neuronal activity, are related to A␤ synthesis and release into the extracellular space (5-7). Under normal physiological conditions and in AD patients, the concentration of A␤ in brain extracellular fluid (interstitial fluid, ISF and cerebrospinal fluid, CSF) is low (10 Ϫ10 M-10 Ϫ9 M) (8...

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...

Huntington disease is caused by mutational expansion of the CAG trinucleotide within exon 1 of the huntingtin (Htt) gene. Exon 1 spanning N-terminal fragments (NTFs) of the Htt protein result from aberrant splicing of transcripts of mutant Htt. NTFs typically encompass a polyglutamine tract flanked by an N-terminal 17-residue amphipathic stretch (N17) and a C-terminal 38-residue proline-rich stretch (C38). We present results from in vitro biophysical studies that quantify the driving forces for and mechanisms of polyglutamine aggregation as modulated by N17 and C38. Although N17 is highly soluble by itself, it lowers the saturation concentration of soluble NTFs and increases the driving force, vis-à-vis homopolymeric polyglutamine, for forming insoluble aggregates. Kinetically, N17 accelerates fibril formation and destabilizes nonfibrillar intermediates. C38 is also highly soluble by itself, and it lends its high intrinsic solubility to lower the driving force for forming insoluble aggregates by increasing the saturation concentration of soluble NTFs. In NTFs with both modules, N17 and C38 act synergistically to destabilize nonfibrillar intermediates (N17 effect) and lower the driving force for forming insoluble aggregates (C38 effect). Morphological studies show that N17 and C38 promote the formation of ordered fibrils by NTFs. Homopolymeric polyglutamine forms a mixture of amorphous aggregates and fibrils, and its aggregation mechanisms involve early formation of heterogeneous distributions of nonfibrillar species. We propose that N17 and C38 act as gatekeepers that control the intrinsic heterogeneities of polyglutamine aggregation. This provides a biophysical explanation for the modulation of in vivo NTF toxicities by N17 and C38. (2). The Htt gene with expanded CAG tracts can undergo erroneous splicing, and the resultant aberrant messenger RNA is translated into a mutant exon 1 version of Htt that is similar to toxic NTFs found in neuronal intranuclear inclusions (3). Exon 1 spanning NTFs typically include a polyglutamine tract that is flanked on its N terminus by an amphipathic 17-residue stretch (MATLEKLMKAFESLKSF) denoted as N17 and by a 38-residue proline-rich stretch on its C terminus (P 11 -QLPQPPPQAQPLLPQPQ-P 10 ) denoted as C38. The N17 sequence is conserved among higher mammals (SI Appendix, Fig. S1), and mutations within N17 impact the properties of NTFs (4, 5). N17 enhances the overall rate of aggregation, as measured by the rate of forming large insoluble species both in vitro (6) and in yeast (7). The C-terminal proline-rich region of exon 1 modulates polyglutamine aggregation and reduces the cellular toxicity of Htt exon 1 even when the polyglutamine tract is significantly expanded (8, 9).A molecular-level understanding of the synergy between the length of polyglutamine tracts and its flanking sequences is essential for inferring the roles of N17 and C38 in vivo. This requires a quantitative understanding of the driving forces, mechanisms, and morphologies for homopolymeric polyglutamine and t...

SummaryPolyglutamine expansions within different proteins are associated with nine different neurodegenerative diseases. There is growing interest in understanding the roles of flanking sequences from disease-relevant proteins on the intrinsic conformational and aggregation properties of polyglutamine. We report results, from atomistic simulations and circular dichroism experiments that quantify the effect of the N-terminal 17-residue (Nt17) segment of the huntingtin protein on polyglutamine conformations and intermolecular interactions. We show that the Nt17 segment and polyglutamine domains become increasingly disordered as polyglutamine length (N) increases in Nt17-Q N constructs. Hydrophobic groups within Nt17 become sequestered in intramolecular, interdomain interfaces. We also show that the Nt17 segment suppresses the intrinsic propensity of polyglutamine aggregation. This inhibition arises from the incipient micellar structures adopted by monomeric forms of the peptides with Nt17 segments. The degree of intermolecular association increases with increasing polyglutamine length and is governed mainly by associations between polyglutamine domains. Comparative analysis of intermolecular associations for different polyglutamine containing constructs leads to clearer interpretations of recently published experimental data. Our results suggest a framework for fibril formation, and identify roles for flanking sequences in modulating polyglutamine aggregation.