Multiscale Molecular Dynamics Studies Reveal Different Modes of Receptor Clustering by Gb3-Binding Lectins.

The recognition of carbohydrate receptors on host cell membranes by pathogenic lectins is a crucial step in the microbial invasion. Two bacterial lectins, the B-subunit of Shiga toxin from Shigella dysenteria (StxB) and lectin I from Pseudomonas aeruginosa (LecA), are specific to the same galactolipid-globotriaosylceramide (Gb3). In this study we present a coarse-grained (cg) model of Gb3, which we further apply to unravel the molecular details of glycolipid binding by two lectins on the surface of a DOPC/cholesterol/Gb3 bilayer. In cg molecular dynamics simulations with time scales of dozens of microseconds, Gb3 was randomly distributed. The binding of both StxB or LecA is accompanied by Gb3 clustering in a cholesterol environment and with exclusion of DOPC in protein vicinity. StxB being bound by all 15 binding sites induced membrane bending, while LecA interacted with two out of four binding sites for most of the time causing a smaller inward curvature of the model membrane. Stable interactions occurred preferably when LecA was normal to the membrane surface. Furthermore, all-atom simulations revealed that LecA bound Gb3's headgroup at only one out of two possible conformations of the carbohydrate moiety observed at protein-free conditions. The results shed light on the mechanism of interactions between two lectins and Gb3 on the membrane surface and offer a coarse-grained model to study more complex systems at large spatiotemporal scales.

Binding of SV40's Viral Capsid Protein VP1 to Its Glycosphingolipid Receptor GM1 Induces Negative Membrane Curvature: A Molecular Dynamics Study.

The binding of the pentameric capsid protein VP1 of simian virus 40 to its glycosphingolipid receptor GM1 is a key step for the entry of the virus into the host cell. Recent experimental studies have shown that the interaction of variants of soluble VP1 pentamers with giant unilamellar vesicles composed of GM1, DOPC, and cholesterol leads to the formation of tubular membrane invaginations to the inside of the vesicles, mimicking the initial steps of endocytosis. We have used coarse-grained and atomistic molecular dynamics (MD) simulations to study the interaction of VP1 with GM1/DOPC/cholesterol bilayers. In the presence of one VP1 protein, we monitor the formation of small local negative curvature and membrane thinning at the protein binding site as well as reduction of area per lipid. These membrane deformations are also observed under cholesterol-free conditions. However, here, the number of GM1 molecules attached to the VP1 binding pockets increases. The membrane curvature is slightly increased for asymmetric GM1 distribution that mimics conditions in vivo, compared to symmetric GM1 distributions which are often applied in experiments. Slightly smaller inward curvature was observed in atomistic control simulations. Binding of four VP1 proteins leads to an increase of the average intrinsic area per lipid in the protein binding leaflet. Membrane fluctuations appear to be the driving force of VP1 aggregation, as was previously shown for membrane-adhering particles because no VP1 aggregation is observed in the absence of a lipid membrane.

Thermodynamic Origin of Multilayer Structures in Langmuir Polymer Films.

The emergence of polymer-free water surface in a Langmuir polymer film at conditions where a homogeneous coverage has been expected previously is explained on the basis of the surface tensions of polymer and water, γpv and γwv, respectively, as well as the interfacial tension between the two materials, γpw. The polymer molecules considered are 22-residue poly(γ-benzyl-l-glutamate) (PBLG) peptides in α-helical conformation. Values for γpv and γpw derived from MD simulations are consistent with values inferred from experiments considering the emergence of polymer-free surface area for ultrathin films studied using the surface forces apparatus in earlier work. Based on these surface properties, the behavior of individual PBLG peptides at the air-water interface, the dimerization of PBLG peptides, the equilibrium height and width of fibers with given cross section, and the lateral fusion of fibers are described. We show that a prerequisite for the emergence of multilayer structures, which appear locally in domains of sizes of tens to hundreds of micrometers in the considered Langmuir polymer film, is that the condition γpv + γpw - γwv > 0 holds true.

Structure Formation in Langmuir Peptide Films As Revealed from Coarse-Grained Molecular Dynamics Simulations.

Molecular dynamics simulations in conjunction with the Martini coarse-grained model have been used to investigate the (nonequilibrium) behavior of helical 22-residue poly(γ-benzyl-l-glutamate) (PBLG) peptides at the water/vapor interface. Preformed PBLG mono- or bilayers homogeneously covering the water surface laterally collapse in tens of nanoseconds, exposing significant proportions of empty water surface. This behavior was also observed in recent AFM experiments at similar areas per monomer, where a complete coverage had been assumed in earlier work. In the simulations, depending on the area per monomer, either elongated clusters or fibrils form, whose heights (together with the portion of empty water surface) increase over time. Peptides tend to align with respect to the fiber axis or with the major principal axis of the cluster, respectively. The aspect ratio of the cluster observed is 1.7 and, hence, comparable to though somewhat smaller than the aspect ratio of the peptides in α-helical conformation, which is 2.2. The heights of the fibrils is 3 nm after 20 ns and increases to 4.5 nm if the relaxation time is increased by 2 orders of magnitude, in agreement with the experiment. Aggregates with heights of about 3 or 4.5 nm are found to correspond to local bi- or trilayer structures, respectively.

Effect of Sodium and Chloride Binding on a Lecithin Bilayer. A Molecular Dynamics Study.

The effect of ion binding on the structural, mechanical, dynamic and electrostatic properties of a 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) bilayer in a 0.5 M aqueous NaCl solution is investigated using classical atomistic molecular dynamics simulation with different force-field descriptions for ion-ion and ion-lipid interactions. Most importantly, the repulsive Lennard-Jones parameters for the latter were modified, such that approximately similar binding of cations and anions to the lipid membrane is achieved. This was done to qualitatively improve the apparent ion-lipid binding constants obtained from simulations with the original force field (Berger lipids and GROMOS87 ions in combination with the SPC water model) in comparison to experimental data. Furthermore, various parameters characterizing membrane structure, elasticity, order and dynamics are analyzed. It is found that ion binding as observed in simulations involving the modified in comparison to the original force-field description leads to: (i) a smaller salt-induced change in the area per lipid, which is in closer agreement with the experiment; (ii) a decrease in the area compressibility and bilayer thickness to values comparable to a bilayer in pure water; (iii) lipid deuterium order parameters and lipid diffusion coefficients on nanosecond timescales that are very similar to the values for a membrane in pure water. In general, salt effects on the structural properties of a POPC bilayer in an aqueous sodium-chloride solution appear to be reproduced reasonably well by the new force-field description. An analysis of membrane-membrane disjoining pressure suggests that the smaller salt-induced change in area per lipid induced by the new force-field description is not due to the alteration of membrane-associated net charge, but must rather be understood as a consequence of ion-specific effects on the arrangement of lipid molecules.

Mechanisms for allosteric activation of protease DegS by ligand binding and oligomerization as revealed from molecular dynamics simulations.

A local perturbation of a protein may lead to functional changes at some distal site, a phenomenon denoted as allostery. Here, we study the allosteric control of a protease using molecular dynamics simulations. The system considered is the bacterial protein DegS which includes a protease domain activated on ligand binding to an adjacent PDZ domain. Starting from crystallographic structures of DegS homo-trimers, we perform simulations of the ligand-free and -bound state of DegS at equilibrium. Considering a single protomer only, the trimeric state was mimicked by applying restraints on the residues in contact with other protomers in the DegS trimer. In addition, the bound state was also simulated without any restraints to mimic the monomer. Our results suggest that not only ligand release but also disassembly of a DegS trimer inhibits proteolytic activity. Considering various observables for structural changes, we infer allosteric pathways from the interface with other protomers to the active site. Moreover, we study how ligand release leads to (i) catalytically relevant changes involving residues 199-201 and (ii) a transition from a stretched to a bent conformation for residues 217-219 (which prohibits proper substrate binding). Finally, based on ligand-induced Cα shifts we identify residues in contact with other protomers in the DegS trimer that likely transduce the perturbation from ligand release from a given protomer to adjacent protomers. These residues likely play a key role in the experimentally known effect of ligand release from a protomer on the proteolytic activity of the other protomers. Proteins 2016; 84:1690-1705. © 2016 Wiley Periodicals, Inc.

Anti-Hemagglutinin Antibody Derived Lead Peptides for Inhibitors of Influenza Virus Binding.

Antibodies against spike proteins of influenza are used as a tool for characterization of viruses and therapeutic approaches. However, development, production and quality control of antibodies is expensive and time consuming. To circumvent these difficulties, three peptides were derived from complementarity determining regions of an antibody heavy chain against influenza A spike glycoprotein. Their binding properties were studied experimentally, and by molecular dynamics simulations. Two peptide candidates showed binding to influenza A/Aichi/2/68 H3N2. One of them, termed PeB, with the highest affinity prevented binding to and infection of target cells in the micromolar region without any cytotoxic effect. PeB matches best the conserved receptor binding site of hemagglutinin. PeB bound also to other medical relevant influenza strains, such as human-pathogenic A/California/7/2009 H1N1, and avian-pathogenic A/Mute Swan/Rostock/R901/2006 H7N1. Strategies to improve the affinity and to adapt specificity are discussed and exemplified by a double amino acid substituted peptide, obtained by substitutional analysis. The peptides and their derivatives are of great potential for drug development as well as biosensing.

Long-Range Conformational Response of a PDZ Domain to Ligand Binding and Release: A Molecular Dynamics Study.

The binding of a ligand to a protein may induce long-range structural or dynamical changes in the biomacromolecule even at sites physically well separated from the binding pocket. A system for which such behavior has been widely discussed is the PDZ2 domain of human tyrosine phosphatase 1E. Here, we present results from equilibrium trajectories of the PDZ2 domain in the free and ligand-bound state, as well as nonequilibrium simulations of the relaxation of PDZ2 after removal of its peptide ligand. The study reveals changes in inter-residue contacts, backbone dihedral angles, and C(α) positions upon ligand release. Our findings show a long-range conformational response of the PDZ2 domain to ligand release in the form of a collective shift of the secondary structure elements α2, ß2, ß3, α1-ß4, and the C terminal loop relative to the rest of the protein away from the N-terminus, and a shift of the loops ß2-ß3 and ß1-ß2 in the opposite direction. The shifts lead to conformational changes in the backbone, especially in the ß2-ß3 loop but also in the ß5-α2 and the α2-ß6 loop, and are accompanied by changes of inter-residue contacts mainly within the ß2-ß3 loop as well as between the α2 helix and other segments. The residues showing substantial changes of inter-residue contacts, backbone conformations, or C(α) positions are considered "key residues" for the long-range conformational response of PDZ2. By comparing these residues with various sets of residues highlighted by previous studies of PDZ2, we investigate the statistical correlation of the various approaches. Interestingly, we find a considerable correlation of our findings with several works considering structural changes but no significant correlations with approaches considering energy flow or networks based on inter-residue energies.

Long-range conformational transition of a photoswitchable allosteric protein: molecular dynamics simulation study.

A local perturbation of a protein may lead to functional changes at some distal site. An example is the PDZ2 domain of human tyrosine phosphatase 1E, which shows an allosteric transition upon binding to a peptide ligand. Recently Buchli et al. presented a time-resolved study of this transition by covalently linking an azobenzene photoswitch across the binding groove and using a femtosecond laser pulse that triggers the cis-trans photoisomerization of azobenzene. To aid the interpretation of these experiments, in this work seven microsecond runs of all-atom molecular dynamics simulations each for the wild-type PDZ2 in the ligand-bound and -free state, as well as the photoswitchable protein (PDZ2S) in the cis and trans states of the photoswitch, in explicit water were conducted. First the theoretical model is validated by recalculating the available NMR data from the simulations. By comparing the results for PDZ2 and PDZ2S, it is analyzed to what extent the photoswitch indeed mimics the free-bound transition. A detailed description of the conformational rearrangement following the cis-trans photoisomerization of PDZ2S reveals a series of photoinduced structural changes that propagate from the anchor residues of the photoswitch via intermediate secondary structure segments to the C-terminus of PDZ2S. The changes of the conformational distribution of the C-terminal region is considered as the distal response of the isolated allosteric protein.

The free energy of nanopores in tense membranes.

Membrane nanopores are central players for a range of important cellular membrane remodeling processes as well as membrane rupture. Understanding pore formation in tense membranes requires comprehension of the molecular mechanism of pore formation and the associated free energy change as a function of the membrane tension. Here we propose a scheme to calculate the free energy change associated with the formation of a nanometer sized pore in molecular dynamics simulations as a function of membrane tension, which requires the calculation of only one computationally expensive potential of mean force. We show that membrane elastic theory can be used to estimate the pore formation free energy at different tension values from the free energy change in a relaxed membrane and the area expansion curves of the membranes. We have computed the pore formation free energy for a dipalmitoyl-phosphatidylcholine (DPPC) membrane at two different lateral pressure values, 1 bar and -40 bar, by calculating the potential of mean force acting on the head group of a single lipid molecule. Unrestrained simulations of the closing process confirm that the intermediate states along this reaction coordinate are reasonable and show that hydrophilic indentations spanning half the bilayer connected by a hydrophobic pore segment represent the corresponding high energy transition state. A comparison of the stability of simulated membranes to experiment at high loading rates show that, contrary to expectation, pores form too easily in small simulated membrane patches. This discrepancy originates from a combination of the absence of ions in the simulations and the small membrane size.

Stable polyglutamine dimers can contain ß-hairpins with interdigitated side chains-but not α-helices, ß-nanotubes, ß-pseudohelices, or steric zippers.

A common thread connecting nine fatal neurodegenerative protein aggregation diseases is an abnormally expanded polyglutamine tract found in the respective proteins. Although the structure of this tract in the large mature aggregates is increasingly well described, its structure in the small early aggregates remains largely unknown. As experimental evidence suggests that the most toxic species along the aggregation pathway are the small early ones, developing strategies to alleviate disease pathology calls for understanding the structure of polyglutamine peptides in the early stages of aggregation. Here, we present a criterion, grounded in available experimental data, that allows for using kinetic stability of dimers to assess whether a given polyglutamine conformer can be on the aggregation path. We then demonstrate that this criterion can be assessed using present-day molecular dynamics simulations. We find that although the α-helical conformer of polyglutamine is very stable, dimers of α-helices lack the kinetic stability necessary to support further oligomerization. Dimers of steric zipper, ß-nanotube, and ß-pseudohelix conformers are also too short-lived to initiate aggregation. The ß-hairpin-containing conformers, instead, invariably form very stable dimers when their side chains are interdigitated. Combining these findings with the implications of recent solid-state NMR data on mature fibrils, we propose a possible pathway for the initial stages of polyglutamine aggregation, in which ß-hairpin-containing conformers act as templates for fibril formation.

Free energy of lipid bilayer defects affected by Alzheimer's disease-associated amyloid-ß42 monomers.

Experimental evidence suggests that the amyloid ß-peptide (Aß) associated with Alzheimer's disease strongly disturbs the integrity of lipid bilayers and cell membranes, as a possible origin of the toxicity of this peptide. Here, we have used molecular dynamics simulations to compute the free energy of membrane pores in the presence and absence of Aß. The validation of our approach included the calculation of lipid flip-flop waiting times, which were found to agree well with recent experiments, in contrast with an earlier simulation study that apparently overestimated these waiting times. We find that, compared with peptide-free lipid bilayers, attached Aß42 peptides (i) increase the order parameters of the lipid tails but (ii) decrease the effective width of the hydrophobic region, (iii) reduce the free energy and thus enlarge the density of membrane pores, and (iv) increase the lifetime of pores. A detailed understanding of the interaction of Aß42 with lipid bilayer membranes may assist in the design of therapeutical strategies against Alzheimer's disease.

Allosteric control of kinesin's motor domain by tubulin: a molecular dynamics study.

Molecular motors such as kinesin are essential for many biological processes. These motors have two motor domains, which bind to tubulin filaments, hydrolyze ATP, and transduce the released chemical energy into directed movements. The general principles of this chemomechanical coupling are now well-established but the underlying molecular mechanisms remain elusive because small conformational changes within large proteins are difficult to detect experimentally. Here, we use atomistic molecular dynamics simulations to monitor such changes within a single motor domain of KIF1A, which belongs to the kinesin-3 motor family. The nucleotide binding pocket of this domain can be empty or occupied by ATP or ADP. For these three nucleotide states, we determine the mobility of the backbone of the protein, both in solution and attached to tubulin. Only one subdomain of the motor domain is found to exhibit a strongly increased mobility upon binding to tubulin: the neck linker that presumably acts as a mechanical transmitter to the other motor domain in dimeric kinesin-3 motors. Furthermore, upon binding to tubulin, the neck linker mobility becomes sensitive to the bound nucleotide and is highly increased after phosphate release, which implies undocking of this linker from the core of the motor domain. These simulation results are consistent with experimental data from EPR spectroscopy, FRET, and cryo-electron microscopy. A detailed analysis of our simulation data also reveals that the undocking of the neck linker in the ADP-kinesin-tubulin state arises from allosteric interactions between the nucleotide and tubulin and that the ß-sheet core undergoes a twist both during phosphate release and ATP binding. The computational approach used here can be applied to other motor domains and mechanoenzymes in order to identify allosteric interactions between the subdomains of these proteins.

Common force field thermodynamics of cholesterol.

Four different force fields are examined for dynamic characteristics using cholesterol as a case study. The extent to which various types of internal degrees of freedom become thermodynamically relevant is evaluated by means of principal component analysis. More complex degrees of freedom (angle bending, dihedral rotations) show a trend towards force field independence. Moreover, charge assignments for membrane-embedded compounds are revealed to be critical with significant impact on biological reasoning.

Negligible water surface charge determined using Kelvin probe and total reflection X-ray fluorescence techniques.

The water surface charge has been extensively debated in recent decades. Electrophoretic mobilities of air bubbles in water and disjoining pressures between the surfaces of aqueous films suggest that the surface of water exhibits a significant negative charge. This is commonly attributed to a strong adsorption of hydroxide ions at the interface, though spectroscopic measurements and simulation studies suggest surface depletion of hydroxide ions. Alternatively, the negative surface charge could arise from surface contamination with trace charged surfactants. We have probed the variation in the surface charge of water with pH by measuring surface potentials using the Kelvin probe technique. Independently, the abundance in the interfacial layer of "reporter ions" (Rb(+) and Br(-)), which must be affected by a charged surface, has been monitored using the total reflection X-ray fluorescence (TRXF) technique. Special care was taken to prove the high sensitivity of this technique as well as to avoid surface contaminants. The magnitude of the surface charge was found to be below 1 e per 500 nm(2) (TRXF). No evidence of variations in the surface potential between pH 2-3 and pH 9-12 was detected within the accuracies of the methods (5 mV for Kelvin probe and 2 mV for TRXF). Hence, our findings suggest that the clean water surface exhibits negligible charge in a wide pH range.

Macro- versus microscopic view on the electrokinetics of a water-membrane interface.

Electrophoresis is an experimental method widely used to study electrostatic properties of interfaces. Here, we question the validity of the macroscopic theory for the planar geometry by Helmholtz and Smoluchowski by considering a POPC bilayer in an aqueous solution with 500 mM NaCl, using molecular dynamics simulations. We find that POPC shows positive electrophoretic mobility due to adsorption of sodium ions at the lipid headgroups. The theory assumes that the region in which the water density undergoes a transition from the bulk value to zero (interfacial width) is small compared to the Debye screening length. This separation of length scale is not fullfilled in the present case. Hence, contrasting the theory, we observe that the surface is not sharply defined, continuum hydrodynamics is not applicable, the effective viscosity in the double layer is increased compared to the bulk, and the zeta potential is dominated by the dipole potential. Our results might have widespread implications for interpretation of electrokinetic studies in general.

Importance of polar solvation and configurational entropy for design of antiretroviral drugs targeting HIV-1 protease.

Both KNI-10033 and KNI-10075 are high affinity preclinical HIV-1 protease (PR) inhibitors with affinities in the picomolar range. In this work, the molecular mechanics Poisson-Boltzmann surface area (MM-PBSA) method has been used to investigate the potency of these two HIV-1 PR inhibitors against the wild-type and mutated proteases assuming that potency correlates with the affinity of the drugs for the target protein. The decomposition of the binding free energy reveals the origin of binding affinities or mutation-induced affinity changes. Our calculations indicate that the mutation I50V causes drug resistance against both inhibitors. On the other hand, we predict that the mutant I84V causes drug resistance against KNI-10075 while KNI-10033 is more potent against the I84V mutant compared to wild-type protease. Drug resistance arises mainly from unfavorable shifts in van der Waals interactions and configurational entropy. The latter indicates that neglecting changes in configurational entropy in the computation of relative binding affinities as often done is not appropriate in general. For the bound complex PR(I50V)-KNI-10075, an increased polar solvation free energy also contributes to the drug resistance. The importance of polar solvation free energies is revealed when interactions governing the binding of KNI-10033 or KNI-10075 to the wild-type protease are compared to the inhibitors darunavir or GRL-06579A. Although the contributions from intermolecular electrostatic and van der Waals interactions as well as the nonpolar component of the solvation free energy are more favorable for PR-KNI-10033 or PR-KNI-10075 compared to PR-DRV or PR-GRL-06579A, both KNI-10033 and KNI-10075 show a similar affinity as darunavir and a lower binding affinity relative to GRL-06579A. This is because of the polar solvation free energy which is less unfavorable for darunavir or GRL-06579A relative to KNI-10033 or KNI-10075. The importance of the polar solvation as revealed here highlights that structural inspection alone is not sufficient for identifying the key contributions to binding affinities and affinity changes for the design of drugs but that solvation effects must be taken into account. A detailed understanding of the molecular forces governing binding and drug resistance might assist in the design of new inhibitors against HIV-1 PR variants that are resistant against current drugs.

Specific binding of chloride ions to lipid vesicles and implications at molecular scale.

Biological membranes composed of lipids and proteins are in contact with electrolytes like aqueous NaCl solutions. Based on molecular dynamics studies it is widely believed that Na(+) ions specifically bind to 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) membranes, whereas Cl(-) ions stay in solution. Here, we present a careful comparison of recent data from electrophoresis and isothermal titration calorimetry experiments as well as molecular dynamics simulations suggesting that in fact both ions show very similar affinities. The corresponding binding constants are 0.44(±0.05) M(-1) for Na(+) and 0.40(±0.04) M(-1) for Cl(-) ions. This is highlighted by our observation that a widely used simulation setup showing asymmetric affinities of Na(+) and Cl(-) for POPC bilayers overestimates the effect of NaCl on the electrophoretic mobility of a POPC membrane by an order of magnitude. Implications for previous simulation results on the effect of NaCl on polarization of interfacial water, transmembrane potentials, and mechanisms for ion transport through bilayers are discussed. Our findings suggest that a range of published simulations results on the interaction of NaCl with phosphocholine bilayers have to be reconsidered and revised and that force field refinements are necessary for reliable simulation studies of membranes at physiological conditions on a molecular level.

Effect of tension and curvature on the chemical potential of lipids in lipid aggregates.

Understanding the factors that influence the free energy of lipids in bilayer membranes is an essential step toward understanding exchange processes of lipids between membranes. In general, both lipid composition and membrane geometry can affect lipid exchange rates between bilayer membranes. Here, the free energy change ΔG(des) for the desorption of dipalmitoyl-phosphatidylcholine (DPPC) lipids from different lipid aggregates has been computed using molecular dynamics simulations and umbrella sampling. The value of ΔG(des) is found to depend strongly on the local properties of the aggregate, in that both tension and curvature lead to an increase in ΔG(des). A detailed analysis shows that the increased desorption free energy for tense bilayers arises from the increased conformational entropy of the lipid tails, which reduces the favorable component -TΔS(L) of the desorption free energy.

Assessing polyglutamine conformation in the nucleating event by molecular dynamics simulations.

Polyglutamine (polyQ) diseases comprise a group of dominantly inherited pathology caused by an expansion of an unstable polyQ stretch which is presumed to form ß-sheets. Similar to other amyloid pathologies, polyQ amyloidogenesis occurs via a nucleated polymerization mechanism, and proceeds through energetically unfavorable nucleus whose existence and structure are difficult to detect. Here, we use atomistic molecular dynamics simulations in explicit solvent to assess the conformation of the polyQ stretch in the nucleus that initiates polyQ fibrillization. Comparison of the kinetic stability of various structures of polyQ peptide with a Q-length in the pathological range (Q40) revealed that steric zipper or nanotube-like structures (ß-nanotube or ß-pseudohelix) are not kinetically stable enough to serve as a template to initiate polyQ fibrillization as opposed to ß-hairpin-based (ß-sheet and ß-sheetstack) or α-helical conformations. The selection of different structures of the polyQ stretch in the aggregation-initiating event may provide an alternative explanation for polyQ aggregate polymorphism.