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)