Infrared spectral frequencies of amide vibrational modes are sensitive to secondary structure. In this work, evidence is presented that accessibility to water additionally affects spectral positions. The dimeric α-helical coiled-coil GCN4-P1‘ was 13C labeled in the amide carbonyl groups of buried Leu or exposed Ala. At 20 °C, the amide I‘ peak for 13C Ala amide is at 1585 cm-1, whereas the position for 13C Leu is at 1606 cm-1. These shifts permit the distinction of solvent-exposed and buried amide groups. Lowering temperature increases H-bond strength, producing a shift to lower frequency. In the temperature range from 10 to 273 K in aqueous glycerol, the amide transitions assigned to solvent-exposed regions of the helices undergo the strongest temperature-dependent shifts, similar to that of the peptide bond model compound, N-methylacetamide, in the same aqueous solvent. In addition, spectral shifts of the amide bands for N-methylacetamide and the solvent-exposed component of the proteins follow the glass transition temperature of the cryosolvent. In contrast, the amide transitions assigned to α-helical segments that are expected to have little interaction with water undergo the weakest shifts. The amide I‘ band of the α-helical protein parvalbumin also shows subpeaks that shift differently with temperature, and on the basis of their temperature dependence and frequency can be assigned to solvent exposed or buried regions. The spectral shifts are discussed in terms of changes in hydrogen bond strengths, including contributions from volume expansion of the sample, and variations in the average hydrogen bond angle, induced by population of low-frequency librational modes involving the solvent and protein. The results on the isotopically labeled peptides conclusively show that α-helical regions that are or are not solvent exposed can be distinguished both by the position of the amide I‘ peak and by the temperature-dependent shifts.
A combination of simulations and Fourier transform infrared spectroscopy was used to examine the effect of three ionic solutes ͑KCl, NaCl, and KSCN͒, the polar solute urea, and the osmolyte trimethylamine-N-oxide ͑TMAO͒ on a water structure. The ionic solutes increase the mean waterwater H-bond angle in their first hydration shell concomitantly shifting the OH stretching mode to higher frequency, and shifting the HOH bending mode to lower frequency. TMAO decreases the mean water-water H-bond angle in its first hydration shell, shifts the OH stretching mode frequency down, and shifting the HOH bending mode frequency up. Urea has no effect on the mean H-bond angle, OH stretch, and HOH bend frequencies. These results can be explained in terms of changes in the relative proportions of two H-bond angle populations: Ionic solutes increase the population of more distorted ͑larger angle͒ H bonds relative to the less distorted population, TMAO has the reverse effect, while urea does not affect the H-bond angle probability distribution. The negligible effect of urea on water structure supports the direct binding model for urea-induced protein denaturation.
We present the structure-based optimization of a series of estrogen receptor-beta (ERbeta) selective ligands. X-ray cocrystal structures of these ligands complexed to both ERalpha and ERbeta are described. We also discuss how molecular modeling was used to take advantage of subtle differences between the two binding cavities in order to optimize selectivity for ERbeta over ERalpha. Quantum chemical calculations are utilized to gain insight into the mechanism of selectivity enhancement. Despite only two relatively conservative residue substitutions in the ligand binding pocket, the most selective compounds have greater than 100-fold selectivity for ERbeta relative to ERalpha when measured using a competitive radioligand binding assay.
We present X-ray crystallographic and molecular modeling studies of estrogen receptors-alpha and -beta complexed with the estrogen receptor-beta-selective phytoestrogen genistein, and coactivator-derived NR box peptides containing an LXXLL motif. We demonstrate that the ligand binding mode is essentially identical when genistein is bound to both isoforms, despite the considerably weaker affinity of this ligand for estrogen receptor-alpha. In addition, we examine subtle differences between binding site residues, providing an explanation for why genistein is modestly selective for the beta isoform. To this end, we also present the results of quantum chemical studies and thermodynamic arguments that yield insight to the nature of the interactions leading to estrogen receptor-beta selectivity. The importance of our analysis to structure-based drug design is discussed.
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