The temperature dependence of the amide I vibrational frequencies of peptides in solution was investigated. In D2O, the amide I' bands of both an alpha-helical oligopeptide, the random-coil poly(L-lysine), and the simplest amide, N-methyl acetamide (NMA), exhibit linear frequency shifts of approximately 0.07 cm(-1)/degrees C with increasing temperature. Similar amide I frequency shifts are also observed for NMA in both polar (acetonitrile and DMSO) and nonpolar (1,4-dioxane) organic solvents, thus ruling out hydrogen-bonding strength as the cause of these effects. The experimental NMA amide I frequencies in the organic solvents can be accurately described by a simple theory based on the Onsager reaction field with temperature-dependent solvent dielectric properties and a solute molecular cavity. DFT-level calculations (BPW91/cc-pVDZ) for NMA with an Onsager reaction field confirm the significant contribution of the molecular cavity to the predicted amide I frequencies. Comparison of the computations to experimental data shows that the frequency-dependent response of the reaction field, taken into account by the index of refraction, is crucial for describing the amide I frequencies in polar solvents. The poor predictions of the model for the NMA amide I band in D2O might be due, in part, to the unknown temperature dependence of the refractive index of D2O in the mid-IR range, which was approximated by the available values in the visible region.

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Infrared (IR) amide I' spectra are widely used for investigations of the structural properties of proteins in aqueous solution. For analysis of the experimental data, it is necessary to separate the spectral features due to the backbone conformation from those arising from other factors, in particular the interaction with solvent. We investigate the effects of solvation on amide I' spectra for a small 40-residue helix-turn-helix protein by theoretical simulations based on density functional theory (DFT). The vibrational force fields and intensity parameters for the protein amide backbone are constructed by transfer from smaller heptaamide fragments; the side chains are neglected in the DFT calculations. Solvent is modeled at two different levels: first as explicit water hydrogen bonded to the surface amide groups, treated at the same DFT level, and, second, using the electrostatic map approach combined with molecular dynamics (MD) simulation. Motional narrowing of the spectral band shapes due to averaging over the fast solvent fluctuation is introduced by use of the time-averaging approximation (TAA). The simulations are compared with the experimental amide I', including two (13)C isotopically edited spectra, corrected for the side-chain signals. Both solvent models are consistent with the asymmetric experimental band shape, which arises from the differential solvation of the amide backbone. However, the effects of (13)C isotopic labeling are best captured by the gas-phase calculations. The limitations of the solvent models and implications for the theoretical simulations of protein amide vibrational spectra are discussed.

The thermal unfolding of a 40-residue helix-turn-helix subdomain of the P22 viral coat protein was investigated using circular dichroism (CD) and Fourier transform infrared spectroscopy (FTIR) with site-specific 13C isotopic labeling. Helix-turn-helix is the simplest alpha-helical structural motif that combines both secondary and tertiary structural elements. The CD of individual helical fragments reveals that the P22 subdomain is stabilized by tertiary interhelical interactions. Overall the temperature-dependent CD and FTIR data can be described by a three-state process with a partially folded intermediate. However, the analysis of the site-specific 13C IR signals reveals distinct unfolding thermodynamics for each of the labeled sites. The thermodynamic parameters of the thermal unfolding of each of the labeled segments were obtained using singular value decomposition in combination with target transformation and global fitting. The P22 subdomain unfolds from the N-terminus toward the helical segments near the turn. Our results show that as few as two 13C labeled residues can be detected in a 40 residue protein and provide local, site-specific structural information about protein unfolding, which is not resolved by standard, nonsite-specific spectroscopic probes.

Infrared (IR) spectroscopy is widely used for studies of temperature-dependent properties of liquids and solutions, such as thermal denaturation of proteins and other molecules of biological interest. The variation of the spectroscopic signals with temperature can be affected by the changes in the optical path length due to the thermal expansion of the components of the sample cell. In this report we investigate the temperature dependence of the optical path length for a liquid IR sample cell of a design typical for aqueous solution experiments. The path lengths were measured from the interference fringes, both in dry cells and with cells partially filled with water. We found that the optical path length variations are significant, on the order of several percent within the temperature range used (0-87 °C). Several commercially available spacers (Teflon, mylar, and lead) and gaskets (Teflon, lead, silicone rubber, Viton, and neoprene) were tested to find materials with either the smallest or most reproducible effect. Teflon, due to its phase transition (known as the "knee point") near room temperature, leads to abrupt changes in path length when used as either spacer or gasket component. On the other hand, Teflon is preferred for its inertness, while several of the other tested materials, most notably lead, are not practically usable due to adhesion to the cell windows upon heating and contact with the aqueous sample. The combination that yielded the most reproducible results, with minimal complications due to adhesion, was Teflon spacer with neoprene gaskets. The implications of the optical path length changes for the temperature-dependent IR experiments and their possible corrections are discussed.