Riley J. Workman

Polyglutamine Fibrils: New Insights into Antiparallel β-Sheet Conformational Preference and Side Chain Structure

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 Huntingtons 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 β-sheet conformations; however, a small population of peptide bonds exist in parallel β-sheet structures. Using MD, we simulate three different potential fibril structural models that consist of either β-strands or β-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 β-strand structure. Importantly, we determine from our MD simulations that Q10 fibril antiparallel β-sheets are thermodynamically more stable than parallel β-sheets. This accounts for why polyQ fibrils preferentially adopt antiparallel β-sheet conformations instead of in-register parallel β-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.

Monomeric Polyglutamine Structures That Evolve into Fibrils

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 β-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 β-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 β-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.

Interaction Enthalpy of Side Chain and Backbone Amides in Polyglutamine Solution Monomers and Fibrils

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.

New Insights into Quinine–DNA Binding Using Raman Spectroscopy and Molecular Dynamics Simulations

Quinines 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 quinines 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 quinines 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.

Assessing Polyglutamine Conformation and Aggregation with Molecular Dynamics Techniques

Huntingtons disease is one of nine neurodegenerative diseases characterized by gene mutations causing polyglutamine (polyQ) repeats in various proteins. Mutated proteins misfold, aggregate, and form amyloid-like fibrils in the neuron. As of now, the aggregation mechanism of these polyglutamine proteins is not well understood. Experimental techniques such as resonance Raman, circular dichroism, and ssNMR are used to analyze properties of polyglutamine solutions. Computational analysis is used in concert with experiment to allow investigation on the molecular level. In this work, short polyQ peptides are studied using molecular dynamics (MD) methods in order to better understand the mechanics of their aggregation. In prior work, we characterized the monomeric conformational ensemble of D2Q10K2 peptides. Our next step is to investigate dimerization properties of these peptides. Adaptive biasing force paired with MD is used to evaluate the dimerization free energies and conformations of D2Q10K2 peptides. On a larger scale, classical MD is used to evaluate the properties of multiple aggregate conformations, including facially stacked β-sheet and β-hairpin sheet systems. Ψ angle probability distributions will be generated from the resulting aggregate trajectories for comparison with experimental distributions. Results from both projects will be presented.

The Conformational Energy Landscape of Aqueous Polyglutamine Peptides from Metadynamics Calculations

One pathological effect of polyglutamine (polyQ) diseases, such as Huntingtons disease, is the aggregation of polyQ tracts in nerve cells. These aggregates form larger fibrils in the cells called inclusions. Though the exact pathological role these inclusions play is unknown, an increase in their development is directly correlated to progression of these diseases. The solution structure and aggregation mechanism of polyQ aggregates is poorly understood and, consequently, is a subject of interest in the biophysics community. Understanding the conformational stability and dynamic properties of polyQ peptides is an important component along the path towards pathological polyQ aggregation. Metadynamics simulation was used to explore the energy landscape of DD(Q)17KK peptides in vacuum, implicit and explicit solvent environments. Initial results from the implicit solvent simulations yield an energy landscape populated by extended and hairpin structures. The results from all simulations will be presented.