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.

We describe the incorporation of a bicyclo[1.1.1]pentane moiety within two known LpPLA inhibitors to act as bioisosteric phenyl replacements. An efficient synthesis to the target compounds was enabled with a dichlorocarbene insertion into a bicyclo[1.1.0]butane system being the key transformation. Potency, physicochemical, and X-ray crystallographic data were obtained to compare the known inhibitors to their bioisosteric counterparts, which showed the isostere was well tolerated and positively impacted on the physicochemical profile.

The 2-phenylnaphthalene scaffold was explored as a simplified version of genistein in order to identify ER selective ligands. With the aid of docking studies, positions 1, 4, and 8 of the 2-phenylnaphthalene template were predicted to be the most potentially influential positions to enhance ER selectivity using two different binding orientations. Both orientations have the phenol moiety mimicking the A-ring of genistein. Several compounds predicted to adopt orientations similar to that of genistein when bound to ERbeta were observed to have slightly higher ER affinity and selectivity than genistein. The second orientation we exploited, which was different from that of genistein when bound to ERbeta, resulted in the discovery of several compounds that had superior ER selectivity and affinity versus genistein. X-ray structures of two ER selective compounds (i.e., 15 and 47) confirmed the alternate binding mode and suggested that substituents at positions 1 and 8 were responsible for inducing selectivity. One compound (i.e., 47, WAY-202196) was further examined and found to be effective in two models of inflammation, suggesting that targeting ER may be therapeutically useful in treating certain chronic inflammatory diseases.

Low temperature UV-visible spectra of cytochrome c and microperoxidase-11 are studied experimentally and theoretically using quantum chemical and Poisson−Boltzmann electrostatics models. Spectral splitting in the Q(0,0) visible absorption band is observed at low temperature (<180 K) in all cytochromes c studied. The Q-band is also found to be blue-shifted with decreasing temperature for cytochrome c and microperoxidase-11. Variations in the energy and splitting of the Q-band are interpreted in terms of heme distortions and interactions of the heme charge distribution with the internal electric field of the heme pocket, generated by charged and polar groups in the protein. The temperature dependence of the spectra is interpreted in terms of coupling of the heme electronic transitions to low frequency vibrational modes and thermal expansion/contraction of the protein−solvent lattice.

Low-temperature UV-vis absorption and Stark-effect hole-burning spectra of Zn substituted cytochrome c are studied experimentally and theoretically using quantum mechanical and Poisson-Boltzmann electrostatics models. Both the Q and Soret bands show resolved splitting at temperatures below ∼180 K. The trend observed in the splittings when comparing cytochromes from different species is found to be the same as that observed for the Q(0,0) band of ferrous cytochrome c. The relative magnitudes of the Q and Soret splittings are found to be consistent with predictions based on Gouterman's four orbital model. For horse heart and yeast cytochrome c, which show the greatest difference in the UV-visible band splittings, Stark effect measurements on persistent spectral holes in the Q(0,0) band indicate that the protein-induced polarization is distinctly different for these two species. Incorporation of the protein electrostatic field as virtual point charges into quantum mechanical calculations utilizing the INDO/s semiempirical Hamiltonian is used to demonstrate that the effects of the protein on the heme electronic structure can be considerably different for the two proteins, consistent with the experimental observations.