RÉSUMÉ
Diffusion determines the turnover of biomolecules in liquid-liquid phase-separated condensates. We considered the mean square displacement and thus the diffusion constant for simple model systems of peptides GGGGG, GGQGG, and GGVGG in aqueous solutions after phase separation by simulating atomic-level models. These solutions readily separate into aqueous and peptide-rich droplet phases. We noted the effect of the peptides being in a solvated, surface, or droplet state on the peptide's diffusion coefficients. Both sequence and peptide conformational distribution were found to influence diffusion and condensate turnover in these systems, with sequence dominating the magnitude of the differences. We found that the most compact structures for each sequence diffused the fastest in the peptide-rich condensate phase. This model result may have implications for turnover dynamics in signaling systems.
Sujet(s)
Condensats biomoléculaires , Peptides , Diffusion , Peptides/composition chimique , Peptides/métabolisme , Condensats biomoléculaires/composition chimique , Condensats biomoléculaires/métabolisme , Séquence d'acides aminés , Eau/composition chimique , Modèles moléculaires , Conformation des protéinesRÉSUMÉ
We present structural models for three different amyloid fibril polymorphs prepared from amylin20-29 (sequence SNNFGAILSS) and amyloid-ß25-35 (Aß25-35) (sequence GSNKGAIIGLM) peptides. These models are based on the amide C=O bond and Ramachandran ψ-dihedral angle data from Raman spectroscopy, which were used as structural constraints to guide molecular dynamics (MD) simulations. The resulting structural models indicate that the basic structural motif of amylin20-29 and Aß25-35 fibrils is extended ß-strands. Our data indicate that amylin20-29 forms both antiparallel and parallel ß-sheet fibril polymorphs, while Aß25-35 forms a parallel ß-sheet fibril structure. Overall, our work lays the foundation for using Raman spectroscopy in conjunction with MD simulations to determine detailed molecular-level structural models of amyloid fibrils in a manner that complements gold-standard techniques, such as solid-state nuclear magnetic resonance and cryogenic electron microscopy.
Sujet(s)
Amyloïde , Analyse spectrale Raman , Amyloïde/composition chimique , Peptides bêta-amyloïdes/composition chimique , Fragments peptidiques/composition chimique , Spectroscopie par résonance magnétique/méthodes , Simulation de dynamique moléculaire , AmidesRÉSUMÉ
Liquid-liquid phase separation of proteins preferentially involves intrinsically disordered proteins or disordered regions. Understanding the solution chemistry of these phase separations is key to learning how to quantify and manipulate systems that involve such processes. Here, we investigate the effect of cyclization on the liquid-liquid phase separation of short polyglycine peptides. We simulated separate aqueous systems of supersaturated cyclic and linear GGGGG and observed spontaneous liquid-liquid phase separation in each of the solutions. The cyclic GGGGG phase separates less robustly than linear GGGGG and has a higher aqueous solubility, even though linear GGGGG has a more favorable single molecule solvation free energy. The versatile and abundant interpeptide contacts formed by the linear GGGGG stabilize the condensed droplet phase, driving the phase separation in this system. In particular, we find that van der Waals close contact interactions are enriched in the droplet phase as opposed to electrostatic interactions. An analysis of the change in backbone conformational entropy that accompanies the phase transition revealed that cyclic peptides lose significantly less entropy in this process as expected. However, we find that the enhanced interaction enthalpy of linear GGGGG in the droplet phase is enough to compensate for a larger decrease in conformational entropy.
Sujet(s)
Protéines intrinsèquement désordonnées , Peptides , Solubilité , Peptides/composition chimique , Entropie , Thermodynamique , Eau/composition chimiqueRÉSUMÉ
Liquid-liquid phase separation of proteins often incorporates intrinsically disordered proteins or those with disordered regions. Examining these processes via the entropy change is desirable for establishing a quantitative foundation with which to probe and understand these phase transitions. Of interest is the effect of residue sequence on the entropy of the peptide backbone. In this work we model these systems via all atom simulations of liquid-liquid phase separation of peptides. Systems of supersaturated pentapeptides separate into a peptide-dense liquid droplet phase as well as a dilute (saturated) aqueous phase. An analysis of the change in backbone conformational entropy associated with the phase transition was performed. We examined systems of four different pentapeptides (GGGGG, GGQGG, GGNGG, and GGVGG) in order to explore the effect of sequence variation on the conformational entropy, as well as the effect of side chain variation on the physical characteristics of the droplet phases. We find that the loss of conformational entropy that accompanies aqueous â droplet transitions is more than compensated by a decrease in interaction enthalpy as contributions to the free energy change for the process.
Sujet(s)
Protéines intrinsèquement désordonnées , Peptides , Entropie , Transition de phase , ThermodynamiqueRÉSUMÉ
Expanded polyglutamine (polyQ) tracts in proteins, which are known to induce their aggregation, are associated with numerous neurodegenerative diseases. Longer polyQ tracts correlate with faster protein aggregation kinetics and a decreased age of onset for polyQ disease symptoms. Here, we use UV resonance Raman spectroscopy, circular dichroism spectroscopy, and metadynamics simulations to investigate the solution-state structures of the D2Q15K2 (Q15) and D2Q20K2 (Q20) peptides. Using metadynamics, we explore the conformational energy landscapes of Q15 and Q20 and investigate the relative energies and activation barriers between these low-energy structures. We compare the solution-state structures of D2Q10K2 (Q10), Q15, and Q20 to determine the dependence of polyQ structure on the Q tract length. We show that these peptides can adopt two distinct monomeric conformations: an aggregation-resistant PPII-like conformation and an aggregation-prone ß-strand-like conformation. We find that longer polyQ peptides have an increased preference for the aggregation-prone ß-strand-like conformation. This preference may play an important role in the increased aggregation rate of longer polyQ peptides that is thought to lead to decreased neurodegenerative disease age of onset for polyQ disease patients.
Sujet(s)
Peptides/composition chimique , Cinétique , Simulation de dynamique moléculaire , Structure en brin bêta , Multimérisation de protéines , ThermodynamiqueRÉSUMÉ
Quinine's ability to bind DNA and potentially inhibit transcription and translation has been examined as a mode of action for its antimalarial activity. UV absorption and fluorescence-based studies have lacked the chemical specificity to develop an unambiguous molecular-level picture of the binding interaction. To address this, we use Raman spectroscopy and molecular dynamics (MD) to investigate quinine-DNA interactions. We demonstrate that quinine's strongest Raman band in the fingerprint region, which derives from a symmetric stretching mode of the quinoline ring, is highly sensitive to the local chemical environment and pH. The frequency shifts observed for this mode in solvents of varying polarity can be explained in terms of the Stark effect using a simple Onsager solvation model, indicating that the vibration reports on the local electrostatic environment. However, specific chemical interactions between the quinoline ring and its environment, such as hydrogen bonding and π-stacking, perturb the frequency of this mode in a more complicated but predictable manner. We use this vibration as a spectroscopic probe to investigate the binding interaction between quinine and DNA. We find that, when the quinoline ring is protonated, quinine weakly intercalates into DNA by forming π-stacking interactions with the base pairs. The Raman spectra indicate that quinine can intercalate into DNA with a ratio reaching up to roughly one molecule per 25 base pairs. Our results are confirmed by MD simulations, which also show that the quinoline ring adopts a t-shaped π-stacking geometry with the DNA base pairs, whereas the quinuclidine head group weakly interacts with the phosphate backbone in the minor groove. We expect that the spectral correlations determined here will enable future studies to probe quinine's antimalarial activities, such as disrupting hemozoin biocrystallization, which is hypothesized to be, among other things, one of its primary modes of action against Plasmodium parasites.
Sujet(s)
ADN/composition chimique , Simulation de dynamique moléculaire , Quinine/composition chimique , Liaison hydrogène , Concentration en ions d'hydrogène , Conformation d'acide nucléique , Théorie quantique , Analyse spectrale RamanRÉSUMÉ
We determined an empirical correlation that relates the amide I vibrational band frequencies of the glutamine (Q) side chain to the strength of hydrogen bonding, van der Waals, and Lewis acid-base interactions of its primary amide carbonyl. We used this correlation to determine the Q side chain carbonyl interaction enthalpy (Δ Hint) in monomeric and amyloid-like fibril conformations of D2Q10K2 (Q10). We independently verified these Δ Hint values through molecular dynamics simulations that showed excellent agreement with experiments. We found that side chain-side chain and side chain-peptide backbone interactions in fibrils and monomers are more enthalpically favorable than are Q side chain-water interactions. Q10 fibrils also showed a more favorable Δ Hint for side chain-side chain interactions compared to backbone-backbone interactions. This work experimentally demonstrates that interamide side chain interactions are important in the formation and stabilization of polyQ fibrils.
RÉSUMÉ
We investigate the solution and fibril conformations and structural transitions of the polyglutamine (polyQ) peptide, D2Q10K2 (Q10), by synergistically using UV resonance Raman (UVRR) spectroscopy and molecular dynamics (MD) simulations. We show that Q10 adopts two distinct, monomeric solution conformational states: a collapsed ß-strand and a PPII-like structure that do not readily interconvert. This clearly indicates a high activation barrier in solution that prevents equilibration between these structures. Using metadynamics, we explore the conformational energy landscape of Q10 to investigate the physical origins of this high activation barrier. We develop new insights into the conformations and hydrogen bonding environments of the glutamine side chains in the PPII and ß-strand-like conformations in solution. We also use the secondary structure-inducing cosolvent, acetonitrile, to investigate the conformations present in low dielectric constant solutions with decreased solvent-peptide hydrogen bonding. As the mole fraction of acetonitrile increases, Q10 converts from PPII-like structures into α-helix-like structures and ß-sheet aggregates. Electron microscopy indicates that the aggregates prepared from these acetonitrile-rich solutions show morphologies similar to our previously observed polyQ fibrils. These aggregates redissolve upon the addition of water! These are the first examples of reversible fibril formation. Our monomeric Q10 peptides clearly sample broad regions of their available conformational energy landscape. The work here develops molecular-level insight into monomeric Q10 conformations and investigates the activation barriers between different monomer states and their evolution into fibrils.
Sujet(s)
Amyloïde/composition chimique , Peptides/composition chimique , Microscopie électronique à transmission , Simulation de dynamique moléculaire , Structure secondaire des protéines , Analyse spectrale RamanRÉSUMÉ
Understanding the structure of polyglutamine (polyQ) amyloid-like fibril aggregates is crucial to gaining insights into the etiology of at least ten neurodegenerative disorders, including Huntington's disease. Here, we determine the structure of D2Q10K2 (Q10) fibrils using ultraviolet resonance Raman (UVRR) spectroscopy and molecular dynamics (MD). Using UVRR, we determine the fibril peptide backbone Ψ and glutamine (Gln) side chain χ3 dihedral angles. We find that most of the fibril peptide bonds adopt antiparallel ß-sheet conformations; however, a small population of peptide bonds exist in parallel ß-sheet structures. Using MD, we simulate three different potential fibril structural models that consist of either ß-strands or ß-hairpins. Comparing the experimentally measured Ψ and χ3 angle distributions to those obtained from the MD simulated models, we conclude that the basic structural motif of Q10 fibrils is an extended ß-strand structure. Importantly, we determine from our MD simulations that Q10 fibril antiparallel ß-sheets are thermodynamically more stable than parallel ß-sheets. This accounts for why polyQ fibrils preferentially adopt antiparallel ß-sheet conformations instead of in-register parallel ß-sheets like most amyloidogenic peptides. In addition, we directly determine, for the first time, the structures of Gln side chains. Our structural data give new insights into the role that the Gln side chains play in the stabilization of polyQ fibrils. Finally, our work demonstrates the synergistic power and utility of combining UVRR measurements and MD modeling to determine the structure of amyloid-like fibrils.