Compact Globular Conformation Research Articles

Entropic Mixing of Ring/Linear Polymer Blends.

The topological constraints of nonconcatenated ring polymers force them to form compact loopy globular conformations with much lower entropy than unconstrained ideal rings. The closed-loop structure of ring polymers also enables them to be threaded by linear polymers in ring/linear blends, resulting in less compact ring conformations with higher entropy. This conformational entropy increase promotes mixing rings with linear polymers. Here, using molecular dynamics simulations for bead-spring chains, ring/linear blends are shown to be significantly more miscible than linear/linear blends and that there is an entropic mixing, negative χ, for ring/linear blends compared to linear/linear and ring/ring blends. In analogy with small angle neutron scattering, the static structure function S(q) is measured, and the resulting data are fit to the random phase approximation model to determine χ. In the limit that the two components are the same, χ = 0 for the linear/linear and ring/ring blends as expected, while χ < 0 for the ring/linear blends. With increasing chain stiffness, χ for the ring/linear blends becomes more negative, varying reciprocally with the number of monomers between entanglements. Ring/linear blends are also shown to be more miscible than either ring/ring or linear/linear blends and stay in single phase for a wider range of increasing repulsion between the two components.

Dual pH- and electro-responsive antibiotic-loaded polymeric platforms for effective bacterial detection and elimination

We describe a multi-tasking flexible system that is able to release a wide spectrum antibiotic (levofloxacin, LVX) under electrostimulation and act as a pH sensor for detecting bacterial infections. Combining anodic polymerization with plasma polymerization processes we engineered dual pH- and electro-responsive polymeric systems. Particularly, the manufactured devices consisted on a layer of poly(hydroxymethyl-3,4-ethylenedioxythiophene) (PHEDOT) loaded with the LVX antibiotic and coated with a plasma polymer layer of poly(acrylic acid) (PAA). The PHEDOT acted as conductive and electro-responsive agent, while the PAA provided pH responsiveness, changing from a compact globular conformation in acid environments to an expanded open coil conformation in alkaline environments. The assembly between the PHEDOT layer and the PAA coating affected the electrochemical response of the former, becoming dependent on the pH detected by the latter. The conformational change experienced by the PAA layer as a function of the pH and the redox properties of PHEDOT were leveraged for the electrochemical detection of bacteria growth and for regulating the release of the LVX antibiotic, respectively. The effectiveness of the system as a stimulus-responsive antibiotic carrier and pH sensor was also investigated on strains of Escherichia coli and Streptococcus salivarius.

Folding of Sequence-Controlled Graft Copolymers to Subdomain-Defined Single-Chain Nanoparticles

We developed a methodology, inspired by the folding of proteins, for the precision synthesis of hairy polymer nanoparticles. High-molar mass and narrowly dispersed graft copolymers were synthesized by graft-through ring opening metathesis polymerization, to incorporate a designated number of side chains and dimerizable cinnamic acid groups. Intrachain photodimerization collapsed the backbone and arrested it into a compact globular conformation, resulting in hairy nanoparticles topologically equivalent to a core cross-linked star polymer. The single-chain collapse process translates the molecular information written on the 1D graft copolymer into the 3D globular polymer nanoparticle, like protein folding. Unprecedented control over structural parameters was achieved, including the length, number, and composition of the side chains as well as cross-linking density. Different side chains formed distinct subdomains in the sterically congested nanoparticle state and further self-assembled into micellar aggregates in a selective solvent. Both experimental observations and computational simulations indicated that preorganization of the side chains in the block sequence produces subdomains which primarily follow the backbone length scale, while random sequences showed side chain-dependent scaling. Polymer nanoparticles with discrete multiple subdomains were produced by folding of the ternary block graft copolymers. Drastic differences in the self-assembly behavior of ABC- and ACB-sequenced nanoparticles indicate that the spatial organization of subdomains can be achieved by sequence control.

Hybrid polarizable simulations of a conventional hydrophobic polyelectrolyte. Toward a theoretical tool for green science innovation.

Hybrid modeling approaches based on all-atom force fields to handle a solute and coarse-grained models to account for the solvent are promising numerical tools that can be used to understand the properties of large and multi-components solutions and thus to speed up the development of new industrial products that obey the standard of green and sustainable chemistry. Here, we discuss the ability of a full polarizable hybrid approach coupled to a standard molecular dynamics scheme to model the behavior in the aqueous phase and at infinite dilution conditions of a standard hydrophobic polyelectrolyte polymer whose charge is neutralized by explicit counterions. Beyond the standard picture of a polyelectrolyte behavior governed by an interplay between opposite intra-polyelectrolyte and inter-polyelectrolyte/counterion Coulombic effects, our simulations show the key role played by both intra-solute polarization effects and long range solute/solvent electrostatics to stabilize compact globular conformations of that polyelectrolyte. Our full polarizable hybrid modeling approach is thus a new theoretical tool well suited to be used in digital strategies for accelerating innovation for green science, for instance.

Insight into the Structure of the “Unstructured” Tau Protein

Combining structural proteomics experimental data with computational methods is a powerful tool for protein structure prediction. Here, we apply a recently developed approach for de novo protein structure determination based on the incorporation of short-distance crosslinking data as constraints in discrete molecular dynamics simulations (CL-DMD), for the determination of the conformational ensemble of tau protein in solution. The predicted structures were inagreement with surface modification and long-distance crosslinking data. Tau in solution was found as an ensemble of rather compact globular conformations with distinct topology, inter-residue contacts, and a number of transient secondary-structure elements. Regions important for pathological aggregation consistently were found to contain β strands. The determined structures are compatible with the tau protein in solution being a molten globule at near-ground state with persistent residual structural features which we were able to capture by CL-DMD. The predicted structure may facilitate an understanding of the misfolding and oligomerization pathways of the tau protein.

Conformational ensemble of native α-synuclein in solution as determined by short-distance crosslinking constraint-guided discrete molecular dynamics simulations.

Combining structural proteomics experimental data with computational methods is a powerful tool for protein structure prediction. Here, we apply a recently-developed approach for de novo protein structure determination based on the incorporation of short-distance crosslinking data as constraints in discrete molecular dynamics simulations (CL-DMD) for the determination of conformational ensemble of the intrinsically disordered protein α-synuclein in the solution. The predicted structures were in agreement with hydrogen-deuterium exchange, circular dichroism, surface modification, and long-distance crosslinking data. We found that α-synuclein is present in solution as an ensemble of rather compact globular conformations with distinct topology and inter-residue contacts, which is well-represented by movements of the large loops and formation of few transient secondary structure elements. Non-amyloid component and C-terminal regions were consistently found to contain β-structure elements and hairpins.

Interaction of peptide backbones and transition metal ions: 1. an IM-MS and DFT study of the binding pattern, structure and fragmentation of Pd(II)/Ni(II)-Polyalanine complexes

The interaction between metal ions and peptides have attracted interests because of the important roles of metal ions in many biological processes and the potential application in different fields. The binding patterns of Pd(II)/Ni(II) with polyalanine and the consequent conformational changes as well as the effects on fragmentation have been studied by electrospray ionization ion mobility mass spectrometry (ESI-IM-MS) experiments and density functional theory (DFT). The collision cross section (CCS) values that were measured showed that such complexes tend to adopt compact globular conformations in the case of polyalanines with more than 6 residues, while conformational change was detected in shorter polyalanines with 4 or 5 residues. Collision induced dissociation (CID) experiments revealed a characteristic fragmentation pattern for Pd(II)/Ni(II)-polyalanine complexes of length n in which neutral part Ala4–H2O + M–2H (M = Pd or Ni) and fragment ions [b4+M–2 H]+, [a4+M–2 H]+ were formed in much higher abundance than any other product ions. Density functional theory (DFT) calculations indicated that Pd(II) and Ni(II) preferred to coordinating to the N atoms of the four residues at the N-terminus. Such a binding pattern accelerates fragmentation involving a structure moiety containing four residues and a metal. This analysis suggested a strong structural correlation between Pd(II) and Ni(II) and the four residues located at the N-terminus of polyalanines.

Controlling the gelation temperature of biomimetic polyisocyanides

Thermosensitive polymers show an entropy-driven transition from a well-solvated to a poorly solvated polymer chain, resulting in a more compact globular conformation. The transition at the lower critical solution temperature (LCST) is often sharp, which allows for a wide range of smart material applications. At the LCST, oligo(ethylene glycol)-substituted polyisocyanides (PICs) form soft hydrogels, composed of polymer bundles similar to biological gels, such as actin, fibrin and intermediate filaments. Here, we show that the LCST of PICs strongly depends linearly on the length of the ethylene glycol (EG) tails; every EG group increases the LCST and thus the gelation temperature by nearly 30°C. Using a copolymerisation approach, we demonstrate that we can precisely tailor the gelation temperature between 10°C and 60°C and, consequently, tune the mechanical properties of the PIC gels.

Polysaccharide from Lycium barbarum L. leaves enhances absorption of endogenous calcium, and elevates cecal calcium transport protein levels and serum cytokine levels in rats

A high-mineral polysaccharide (LP) was extracted from Lycium barbarum L. leaves, and the absorption of endogenous calcium, iron, and zinc was evaluated in Caco-2 cells and rat models. The absorption of these minerals ranged from 2.7 to 5.9% in Caco-2 cells, compared with 60.5–81.9% in rat models. LP had a compact globular conformation at pH∼7.2 when co-cultured with Caco-2 cells, and degraded to a loosely linear structure at pH∼6.7 in rat cecum. The degraded polysaccharides didn’t change the mucosal morphometry but caused higher expression of calcium transporter 1 (CAT1) and zinc transporter 1 (ZIP1) proteins in rat cecum. Furthermore, fermentation of polysaccharides in cecum also caused elevated levels of serum cytokines, including TNF-α and IL-6. GL polysaccharides could increase the bioavailability of endogenous calcium via changes in the molecular conformation and through upregulation of the expression of mineral transporter proteins.

Phosphorylation regulates the secondary structure and function of dentin phosphoprotein peptides

Dentin phosphoprotein (DPP) is the most acidic protein in vertebrates and structurally is classified as an intrinsically disordered protein. Functionally, DPP is related to dentin and bone formation, however the specifics of such association remain unknown. Here, we used atomistic molecular dynamics simulations to screen selected binding domains of DPP onto hydroxyapatite (HA), which is one of its important interacting partners. From these results, we selected a functionally relevant peptide, Ace-SSDSSDSSDSSDSSD-NH2 (named P5) and its phosphorylated form (named P5P), for experimental characterization. SAXS experiments indicated that in solution P5 was disordered, possibly in an extended conformation while P5P displayed more compact globular conformations. Circular dichroism and FTIR confirmed that, either in the presence or absence of Ca2+/HA, P5 adopts a random coil structure, whereas its phosphorylated counterpart, P5P, has a more compact arrangement associated with conformations that display β-sheet and α-helix motifs when bound to HA. In solution, P5 inhibited HA crystal growth, whereas at similar concentrations, P5P stimulated it. These findings suggest that phosphorylation controls the transient formation of secondary and tertiary structure of DPP peptides, and, most likely of DPP itself, which in turn controls HA growth in solution and possibly HA growth in mineralized tissues.

On the urea induced hydrophobic collapse of a water soluble polymer.

Stabilization of macromolecular folded states in solution by protective osmolytes has been traditionally explained on the basis of preferential osmolyte depletion from the macromolecule's first solvation shell. However recent theoretical and experimental studies suggest that protective osmolytes may directly interact with the macromolecule. An example is the stabilization of the collapsed globular state of poly(N-isopropylacrylamide) (PNiPAM) by urea in aqueous solution. Based on Molecular Dynamics simulations we have characterized the mechanism through which urea stabilizes the collapsed state of PNiPAM in water. Analysis and comparison of the different components of the excess chemical potentials of folded and unfolded PNiPAM chains in aqueous urea solutions indicates that enthalpic interactions play no role in stabilizing the collapsed state. We instead find that with increasing urea, solvation of the unfolded state is entropically penalized over solvation of the folded state, thereby shifting the folding equilibrium in favour of the folded state. The unfavourable entropy contribution to the excess chemical potential of unfolded PNiPAM chains results from two urea effects: (1) an increasing cost of cavity formation with increasing urea, (2) larger fluctuations in the energy component corresponding to PNiPAM-(co)solvent attractive interactions. These energy fluctuations are particularly relevant at low urea concentrations (<3 M) and result from attractive polymer-urea van der Waals interactions that drive the formation of "urea clouds" but bias the spatial distribution of urea and water molecules with a corresponding reduction of the entropy. We further find indications that urea increases the entropy of the globular state.

Abstract 2891: Comparative analyses of human estrogen receptor- EF-hand protein complexes: molecular basis for hormone independent activation.

Abstract Estrogen Receptor (ER) belongs to the nuclear receptor super family of ligand-triggered transcription factors. ER is present in about 70% of breast cancers and plays a critical role in the biology of breast carcinoma and improves prognosis. Estrogen binds to ER and mediates a cascade of cellular signaling events. It has been shown that several proteins including calcium binding proteins with EF-hand motifs, which control cellular calcium and calcium mediated signaling pathways, including calmodulin (CaM) could bind to unliganded ER and activate ER. CaM has four EF-hand motifs that change conformation upon binding Ca2+ ions. Ca2+-CaM complex binds to the target proteins and initiates various signaling cascades. Each EF-hand motif contains two alpha helices connected by a 12-residue loop. Both X-ray and NMR studies show that the alpha helices of the EF-hand motifs change their position relative to each other, forming an almost perpendicular conformation. This allows calmodulin to increase its binding affinity for a number of target proteins. CaM forms a compact globular conformation by bending its central helix upon binding its target peptides. S100, a soluble protein that is recently reported to interact with ER has only two EF-hand motifs. S100 protein is overexpressed in several cancers and shares a high degree of sequence homology with calmodulin. Methods: Protein-protein interactions of CaM, S100 with ER alpha were simulated using HADDOCK and HEX. Docking results were analyzed in UCSF Chimera and integrated with data from in vitro proteomics experiments to determine the conformations of ER bound EF-hand protein complexes. Proteomic experiments consisted of chemical crosslinking of the corresponding protein complexes followed by tryptic digestion, analyzing the resulting peptide peaks using FTICR- Mass Spectrometry. Results/Discussion: Using the fold information and contact regions obtained from the Mass spectrometry, 3D structures of ER-EF hand protein complexes were reconstructed. These structures of ER best represent the conformational state sensed by the specific interacting partner. ER-S100 complex is distinct from CaM. The lack of the connective peptidic region between the EF-hand pairs in S100 attributed to less interaction coverage indicative of only inducing a partial agonist-like conformation. We believe that the ensemble of ER-EF hand protein complexes generated by our integrated proteomics-assisted protein interaction profiling will shed light on the lingering issue of hormone independent activation of ER at the molecular level. Acknowledgements: This research was supported via funds from the New York State, National Institutes of Health (5P20MD002725), through the NCMHD -RIMI program (RVR) Citation Format: David H. Lee, Bethany K. Asare, Suchitra Singh, Rajendram V. Rajnarayanan. Comparative analyses of human estrogen receptor- EF-hand protein complexes: molecular basis for hormone independent activation. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2891. doi:10.1158/1538-7445.AM2013-2891

Easy synthesis of dendrimer-like polymers through a divergent iterative “end-grafting” method

We report here an easy method for the synthesis of dendrimer-like polymers with high branching functionality (1 → 8). The synthetic process involves iterative grafting reactions in a divergent way. A multi-functional core containing short segments of polyisoprene (PI), either as a star-like block copolymer of isoprene and styrene or as a linear triblock copolymer of isoprene, styrene and isoprene (coded G1), is epoxidized on the double bonds and grafted with a living block copolymer, polyisoprene-b-polystyrenyllithium (PI-b-PSLi), again with a short PI segment, through the ring-opening reaction of oxirane by polymeric anions. The resulting graft polymer, G2, possesses a definite number of PI segments at the periphery. These PI segments are further epoxidized followed by the ring-opening addition of PI-b-PSLi, affording G3. Repeating the process leads to the synthesis of a dendrimer-like polystyrene up to 5th generation with a polydispersity lower than 1.21, as measured by SEC. A feature of the process is the easily accessible high chain density in the final product, although defects exist due to steric hindrance in the reactions of high generations. The solution properties of the dendritic products are investigated using viscometry and dynamic and static laser light scattering on the molecular conformation. The results support a compact globular conformation model for the dendrimer-like products. In addition, the chain density of the products from the star-like core is higher than that of products from a linear triblock core. AFM results show that the dendritic products adopt flattened conformations and tend to form lateral sphere-like aggregates on mica substrate.

A Compact Intermediate State of Calmodulin in the Process of Target Binding

Calmodulin undergoes characteristic conformational changes by binding Ca(2+), which allows it to bind to more than 300 target proteins and regulate numerous intracellular processes in all eukaryotic cells. We measured the conformational changes of calmodulin upon Ca(2+) and mastoparan binding using the time-resolved small-angle X-ray scattering technique combined with flash photolysis of caged calcium. This measurement system covers the time range of 0.5-180 ms. Within 10 ms of the stepwise increase in Ca(2+) concentration, we identified a distinct compact conformational state with a drastically different molecular dimension. This process is too fast to study with a conventional stopped-flow apparatus. The compact conformational state was also observed without mastoparan, indicating that the calmodulin forms a compact globular conformation by itself upon Ca(2+) binding. This new conformational state of calmodulin seems to regulate Ca(2+) binding and conformational changes in the N-terminal domain. On the basis of this finding, an allosteric mechanism, which may have implications in intracellular signal transduction, is proposed.

Conformational Plasticity of proNGF

Nerve Growth Factor is an essential protein that supports neuronal survival during development and influences neuronal function throughout adulthood, both in the central and peripheral nervous system. The unprocessed precursor of NGF, proNGF, seems to be endowed with biological functions distinct from those of the mature protein, such as chaperone-like activities and apoptotic and/or neurotrophic properties. We have previously suggested, based on Small Angle X-ray Scattering data, that recombinant murine proNGF has features typical of an intrinsically unfolded protein. Using complementary biophysical techniques, we show here new evidence that clarifies and widens this hypothesis through a detailed comparison of the structural properties of NGF and proNGF. Our data provide direct information about the dynamic properties of the pro-peptide and indicate that proNGF assumes in solution a compact globular conformation. The N-terminal pro-peptide extension influences the chemical environment of the mature protein and protects the protein from proteolytic digestion. Accordingly, we observe that unfolding of proNGF involves a two-steps mechanism. The distinct structural properties of proNGF as compared to NGF agree with and rationalise a different functional role of the precursor.

Structure and mechanism of calmodulin binding to a signaling sphingolipid reveal new aspects of lipid‐protein interactions

Lipid-protein interactions are rarely characterized at a structural molecular level due to technical difficulties; however, the biological significance of understanding the mechanism of these interactions is outstanding. In this report, we provide mechanistic insight into the inhibitory complex formation of the lipid mediator sphingosylphosphorylcholine with calmodulin, the most central and ubiquitous regulator protein in calcium signaling. We applied crystallographic, thermodynamic, kinetic, and spectroscopic approaches using purified bovine calmodulin and bovine cerebral microsomal fraction to arrive at our conclusions. Here we present 1) a 1.6-Å resolution crystal structure of their complex, in which the sphingolipid occupies the conventional hydrophobic binding site on calmodulin; 2) a peculiar stoichiometry-dependent binding process: at low or high protein-to-lipid ratio calmodulin binds lipid micelles or a few lipid molecules in a compact globular conformation, respectively, and 3) evidence that the sphingolipid displaces calmodulin from its targets on cerebral microsomes. We have ascertained the specificity of the interaction using structurally related lipids as controls. Our observations reveal the structural basis of selective calmodulin inhibition by the sphingolipid. On the basis of the crystallographic and biophysical characterization of the calmodulin-sphingosylphosphorylcholine interaction, we propose a novel lipid-protein binding model, which might be applicable to other interactions as well.

A pH-responsive interface derived from resilin-mimetic protein Rec1- resilin

In this investigation, for the first time we report the effects of pH on the molecular orientation, packing density, structural properties, adsorption characteristics and viscoelastic behaviour of resilin-mimetic protein rec1- resilin at the solid–liquid interface using quartz crystal microbalance with dissipation monitoring (QCM-D) and surface plasmon resonance (SPR) spectroscopy. QCM-D and SPR data confirm that the binding ability of rec1- resilin on a substrate is strongly pH-dependent the protein packing density on a gold surface is calculated to be 4.45 × 10 13 per cm 2 at the isoelectric point (IEP ∼ 4.9), 8.79 × 10 11 per cm 2 at pH 2 and 9.90 × 10 11 per cm 2 at pH 12, respectively. Our findings based on the thickness, dissipation and viscoelasticity of the rec1- resilin adlayer also indicate that it is adsorbed onto the gold substrate with different orientation depending on pH, such as back-on adsorption at acidic pH of 2, compact end-on bilayer adsorption at the IEP and side-on at high alkaline pH of 12. When rec1- resilin is ‘pinned’ to the substrate at IEP and subsequently exposed to an electrolyte solution adjusted to different pH, it switches from a compact globular conformation of the bio-macromolecule at the IEP to a coil conformation at pH between IEP to IED (IED = pKa value of tyrosine amino acid residue) and an extended coil conformation at pH > IED. This transformation from globule to coil to extended coil conformation is kinetically fast, robust and completely reversible. Such responsive surfaces created using ‘smart’ biomimetic rec1- resilin have the potential to find applications in many areas including biotechnology, medicine, sensors, controlled drug delivery systems and engineering.

New Aspects of Lipid-Protein Interactions Revealed by Calmodulin Binding to the Lipid Mediator Sphingosylphosphorylcholine

Lipid-protein interactions drive core cellular signaling events and regulate the function of most membrane proteins. Despite their significance, molecular details of these interactions are scarcely known. Here we provide mechanistic insight into the binding of the lipid mediator sphingosylphosphorylcholine to calmodulin, the most central and ubiquitous regulator protein in calcium signaling, and present a crystal structure of their complex. Thermodynamic and kinetic analysis of their interaction revealed a peculiar stoichiometry-dependent binding process. At low protein-to-lipid ratios, calmodulin binds with high affinity to lipid micelles. At high protein-to-lipid ratios, however, the resulting complex adopts a compact globular conformation with calmodulin embracing a few lipid molecules, as can be seen in our crystal structure. Intriguingly, the sphingolipid occupies the binding pocket for the amphipatic alpha-helices of calmodulin's target proteins. This finding explains the competitive inhibition of calmodulin function by the signaling lipid, and proposes an utterly novel type of endogenous regulation for the calcium sensor protein. The binding model presented here might be widely applicable to other lipid-protein interactions as well.View Large Image | View Hi-Res Image | Download PowerPoint Slide

The Effect of Calcium Ions and Peptide Ligands on the Relative Stabilities of the Calmodulin Dumbbell and Compact Structures

A combination of ion mobility and mass spectrometry methods was used to characterize the molecular shape of the protein calmodulin (CaM) and its complexes with calcium and a number of peptide ligands. CaM, a calcium-binding protein composed of 148 amino acid residues, was found by X-ray crystallography to occur both in a globular shape and in the shape of an extended dumbbell. Here, it was found, as solutions of CaM and CaM complexes were sprayed into the solvent-free environment of the mass spectrometer, that major structural features of the molecule and the stoichiometry of the units constituting a complex in solution were preserved in the desolvation process. Two types of CaM structures were observed in our experiments: a compact and an extended form of CaM with measured cross sections in near-perfect agreement with those calculated for the known globular and extended dumbbell X-ray geometries. Calcium-free solutions yielded predominantly an extended CaM conformation. Ca(n)(2+)-CaM complexes were observed in calcium-containing solutions, n = 0-4, with the population of the compact conformation increasing relative to the elongated conformation as n increases. For n = 4, a predominantly compact globular conformation was observed. Solutions containing the peptide CaMKII(290-309), the CaM target domain of the Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) enzyme, yielded predominantly globular Ca(4)(2+)-CaM-CaMKII(290-309) complexes. Similar results were obtained with the 26-residue peptide melittin. For the 14-residue C-terminal melittin fragment, on the other hand, formation of both a 1:1 and a 1:2 CaM-peptide complex was detected. On the basis of the entirety of our results, we conclude that the collapse of extended (dumbbell-like) CaM structures into more compact globular structures occurs upon specific binding of four calcium ions. Furthermore, this calcium-induced structural collapse of CaM appears to be a prerequisite for formation of a particularly stable CaM-peptide complex involving peptides long enough to be engaged in interactions with both lobes of CaM.

Single homopolypeptide chains collapse into mechanically rigid conformations

Huntington's disease is linked to the insertion of glutamine (Q) in the protein huntingtin, resulting in polyglutamine (polyQ) expansions that self-associate to form aggregates. While polyQ aggregation has been the subject of intense study, a correspondingly thorough understanding of individual polyQ chains is lacking. Here we demonstrate a single molecule force-clamp technique that directly probes the mechanical properties of single polyQ chains. We have made polyQ constructs of varying lengths that span the length range of normal and diseased polyQ expansions. Each polyQ construct is flanked by the I27 titin module, providing a clear mechanical fingerprint of the molecule being pulled. Remarkably, under the application of force, no extension is observed for any of the polyQ constructs. This is in direct contrast with the random coil protein PEVK of titin, which readily extends under force. Our measurements suggest that polyQ chains form mechanically stable collapsed structures. We test this hypothesis by disrupting polyQ chains with insertions of proline residues and find that their mechanical extensibility is sensitive to the position of the proline interruption. These experiments demonstrate that polyQ chains collapse to form a heterogeneous ensemble of conformations that are mechanically resilient. We further use a heat-annealing molecular dynamics protocol to extensively search the conformation space and find that polyQ can exist in highly mechanically stable compact globular conformations. The mechanical rigidity of these collapsed structures may exceed the functional ability of eukaryotic proteasomes, resulting in the accumulation of undigested polyQ sequences in vivo.