Temperature Dependence Of Amide Research Articles

Temperature Dependence of the Amide I Frequency as a Probe of Solvent Accessibility of the Protein Backbone

Infrared (IR) spectroscopy of proteins often focuses on the relationship between the amide I band and polypeptide secondary structure; IR spectra has been used to probe backbone conformation on a wide range of samples (solution, films, gels, and solids), at a broad range of time scales (from ns to days), and as a function of a variety of perturbations (solvent, ionic strength, presence of lipid membrane). Often ignored is the solvent dependence of the amide I mode. Solvent-backbone hydrogen bonding can have a large effect on the observed amide I frequency (1). In this work, we demonstrate that the temperature dependence of amide I bands can be used to determine the solvent exposure of the peptide backbone. We have compared the IR spectra as a function of temperature in a number of different protein systems, including small, alanine-based peptides that form alpha-helices in solution; short peptides that aggregate to form fibrous, beta-sheet rich fibrous structures; short, dynamic peptides lacking regular secondary structure; membrane-embedded peptides; and globular proteins. When backbone groups are buried from solvent, the amide I band frequencies are independent of temperature (in the range of 5°C to 75°C). By contrast, backbone groups exposed to (and hydrogen-bonded with) solvent water show a temperature dependence similar to that observed in solvated model compounds (2). When this approach is combined with specific isotope-labeling (3), site-specific information about solvent accessibility can be obtained from the variable-temperature IR spectra. (1) A. Starzyk, W. Barber-Armstrong, M. Sridharan and S. M. Decatur, Biochemistry, 2005, 44, 369. (2) K. E. Amunson and J. Kubelka J. Phys. Chem. B, 2007, 111, 9993. (3) S. M. Decatur, Acc. Chem. Res., 2006, 39, 169.

On the temperature dependence of amide I intensities of peptides in solution

Temperature dependence of the amide I/I′ spectral intensities is investigated for N-methylacetamide (NMA) as a model compound for the peptide bond in D 2O and three organic solvents with different polarities (dimethyl sulfoxide (DMSO), acetonitrile and 1,4-dioxane). The total amide I/I′ intensity (dipole strength) systematically decreases in less polar solvents as well as with the increasing temperature. Decreased solvent polarity results in the narrowing of the amide I bandwidths, while increasing temperature predominantly reduces the peak absorbance, with only a small effect on the spectral width. In D 2O, the NMA amide I′ dipole strength decreases by 1.7 × 10 −4 Debye 2/deg, in DMSO, acetonitrile and 1,4-dioxane by 1.0 × 10 −4 Debye 2/deg. The amide I/I′ intensity variations in the non-protic solvents rule out hydrogen bonding as the sole source of these effects. The experimental NMA amide I dipole strengths in the organic solvents are accurately described by a simple theory based on the Onsager reaction field with temperature-dependent solvent dielectric constant, refractive index and the solute molecular cavity, which can be approximated using NMA density. Experimental results are compared to density functional theory (DFT) BPW91/cc-pVDZ/Onsager calculations. The computations significantly overestimate the absolute experimental amide I intensities, but comparison of the relative values underscores the importance of the temperature-dependent molecular cavity dimension (density) as well as the frequency-dependent response of the reaction field (index of refraction) for describing the amide I spectral intensities in polar solvents. Correlations between temperature-dependent amide I frequencies and intensities, and their possible utility for analyses of the temperature-dependent peptide and protein infrared spectroscopy (IR) spectra are discussed.

On the Temperature Dependence of Amide I Frequencies of Peptides in Solution

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.