UV Resonance Raman Spectroscopy Monitors Polyglutamine Backbone and Side Chain Hydrogen Bonding and Fibrillization

We utilize 198 and 204 nm excited UV resonance Raman spectroscopy (UVRR) and circular dichroism spectroscopy (CD) to monitor the backbone conformation and the Gln side chain hydrogen bonding (HB) of a short, mainly polyGln peptide with a D₂Q₁₀K₂ sequence (Q10). We measured the UVRR spectra of valeramide to determine the dependence of the primary amide vibrations on amide HB. We observe that a nondisaggregated Q10 (NDQ10) solution (prepared by directly dissolving the original synthesized peptide in pure water) exists in a β-sheet conformation, where the Gln side chains form hydrogen bonds to either the backbone or other Gln side chains. At 60 °C, these solutions readily form amyloid fibrils. We used the polyGln disaggregation protocol of Wetzel et al. [Wetzel, R., et al. (2006) <i>Methods Enzymol.</i> <i>413</i>, 34–74] to dissolve the Q10 β-sheet aggregates. We observe that the disaggregated Q10 (DQ10) solutions adopt PPII-like and 2.5₁-helix conformations where the Gln side chains form hydrogen bonds with water. In contrast, these samples do not form fibrils. The NDQ10 β-sheet solution structure is essentially identical to that found in the NDQ10 solid formed upon evaporation of the solution. The DQ10 PPII and 2.5₁-helix solution structure is essentially identical to that in the DQ10 solid. Although the NDQ10 solution readily forms fibrils when heated, the DQ10 solution does not form fibrils unless seeded with the NDQ10 solution. This result demonstrates very high activation barriers between these solution conformations. The NDQ10 fibril secondary structure is essentially identical to that of the NDQ10 solution, except that the NDQ10 fibril backbone conformational distribution is narrower than in the dissolved species. The NDQ10 fibril Gln side chain geometry is more constrained than when NDQ10 is in solution. The NDQ10 fibril structure is identical to that of the DQ10 fibril seeded by the NDQ10 solution

Les paysans français aux 17ème et 18ème siècles : les campagnes françaises avant la révolution / Michel Adenis, réal. ; Jacques Dupâquier, Janine Codou, aut. ; Robert Party, voix

Collection : HistoireRésumé : À travers les archives et les paysage, c'est en suivant Arthur Young dans son voyage que l'émission fait découvrir aux élèves ce qu'étaient la France rurale à la veille de la Révolution, la diversité des paysages ruraux et des conditions de la vie paysanne. (source : Canopé)Durée : 00:18:55Thème : Histoir

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<i>H</i>_int) in monomeric and amyloid-like fibril conformations of D₂Q₁₀K₂ (Q10). We independently verified these Δ<i>H</i>_int 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<i>H</i>_int 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

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 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 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

2D Photonic Crystal Protein Hydrogel Coulometer for Sensing Serum Albumin Ligand Binding

Bovine and human serum albumin (BSA and HSA) are globular proteins that function as bloodstream carriers of hydrophobes such as fatty acids and drugs. We fabricated novel photonic crystal protein hydrogels by attaching 2D colloidal arrays onto pure BSA and HSA hydrogels. The wavelengths of the diffracted light sensitively report on the protein hydrogel surface area. The binding of charged species to the protein hydrogel gives rise to Donnan potentials that change the hydrogel volume causing shifts in the diffraction. These photonic crystal protein hydrogels act as sensitive Coulometers that monitor the hydrogel charge state. We find multiple high-affinity BSA and HSA binding sites for salicylate, ibuprofen and picosulfate by using these sensors to monitor binding of charged drugs. We demonstrate proof-of-concept for utilizing protein hydrogel sensors to monitor protein–ionic species binding

Glutamine and Asparagine Side Chain Hyperconjugation-Induced Structurally Sensitive Vibrations

We identified vibrational spectral marker bands that sensitively report on the side chain structures of glutamine (Gln) and asparagine (Asn). Density functional theory (DFT) calculations indicate that the Amide III^P (AmIII^P) vibrations of Gln and Asn depend cosinusoidally on their side chain OCCC dihedral angles (the χ₃ and χ₂ angles of Gln and Asn, respectively). We use UV resonance Raman (UVRR) and visible Raman spectroscopy to experimentally correlate the AmIII^P Raman band frequency to the primary amide OCCC dihedral angle. The AmIII^P structural sensitivity derives from the Gln (Asn) C_β–C_γ (C_α–C_β) stretching component of the vibration. The C_β–C_γ (C_α–C_β) bond length inversely correlates with the AmIII^P band frequency. As the C_β–C_γ (C_α–C_β) bond length decreases, its stretching force constant increases, which results in an upshift in the AmIII^P frequency. The C_β–C_γ (C_α–C_β) bond length dependence on the χ₃ (χ₂) dihedral angle results from hyperconjugation between the C_δO_ϵ (C_γO_δ) π* and C_β–C_γ (C_α–C_β) σ orbitals. Using a Protein Data Bank library, we show that the χ₃ and χ₂ dihedral angles of Gln and Asn depend on the peptide backbone Ramachandran angles. We demonstrate that the inhomogeneously broadened AmIII^P band line shapes can be used to calculate the χ₃ and χ₂ angle distributions of peptides. The spectral correlations determined in this study enable important new insights into protein structure in solution, and in Gln- and Asn-rich amyloid-like fibrils and prions

UV Resonance Raman Investigation of the Aqueous Solvation Dependence of Primary Amide Vibrations

We investigated the normal mode composition and the aqueous solvation dependence of the primary amide vibrations of propanamide. Infrared, normal Raman, and UV resonance Raman (UVRR) spectroscopy were applied in conjunction with density functional theory (DFT) to assign the vibrations of crystalline propanamide. We examined the aqueous solvation dependence of the primary amide UVRR bands by measuring spectra in different acetonitrile/water mixtures. As previously observed in the UVRR spectra of <i>N</i>-methylacetamide, all of the resonance enhanced primary amide bands, except for the Amide I (AmI), show increased UVRR cross sections as the solvent becomes water-rich. These spectral trends are rationalized by a model wherein the hydrogen bonding and the high dielectric constant of water stabilizes the <i>ground state</i> dipolar ^–OCNH₂⁺ resonance structure over the neutral OCNH₂ resonance structure. Thus, vibrations with large CN stretching show increased UVRR cross sections because the CN displacement between the electronic ground and excited state increases along the CN bond. In contrast, vibrations dominated by CO stretching, such as the AmI, show a decreased displacement between the electronic ground and excited state, which result in a decreased UVRR cross section upon aqueous solvation. The UVRR primary amide vibrations can be used as sensitive spectroscopic markers to study the local dielectric constant and hydrogen bonding environments of the primary amide side chains of glutamine (Gln) and asparagine (Asn)

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

Aluminum Film-Over-Nanosphere Substrates for Deep-UV Surface-Enhanced Resonance Raman Spectroscopy

We report here the first fabrication of aluminum film-over nanosphere (AlFON) substrates for UV surface-enhanced resonance Raman scattering (UVSERRS) at the deepest UV wavelength used to date (λ_ex = 229 nm). We characterize the AlFONs fabricated with two different support microsphere sizes using localized surface plasmon resonance spectroscopy, electron microscopy, SERRS of adenine, tris­(bipyridine)­ruthenium­(II), and trans-1,2-bis­(4-pyridyl)-ethylene, SERS of 6-mercapto-1-hexanol (as a nonresonant molecule), and dielectric function analysis. We find that AlFONs fabricated with the 210 nm microspheres generate an enhancement factor of approximately 10^4–5, which combined with resonance enhancement of the adsorbates provides enhancement factors greater than 10⁶. These experimental results are supported by theoretical analysis of the dielectric function. Hence our results demonstrate the advantages of using AlFON substrates for deep UVSERRS enhancement and contribute to broadening the SERS application range with tunable and affordable substrates