We examine the structural changes during the primary photoreaction in blue-absorbing proteorhodopsin (BPR), a light-driven retinylidene proton pump, using low-temperature FTIR difference spectroscopy. Comparison of the light-induced BPR difference spectrum recorded at 80 K to that of green-absorbing proteorhodopsin (GPR) reveals that there are several differences in the BPR and GPR primary photoreactions despite the similar structure of the retinal chromophore and all-trans → 13-cis isomerization. Strong bands near 1700 cm⁻¹ assigned previously to a change in hydrogen bonding of Asn230 in GPR are still present in BPR. However, additional bands in the same region are assigned on the basis of site-directed mutagenesis to changes occurring in Gln105. In the amide II region, bands are assigned on the basis of total ¹⁵N labeling to structural changes of the protein backbone, although no such bands were previously observed for GPR. A band at 3642 cm⁻¹ in BPR, assigned to the OH stretching mode of a water molecule on the basis of H₂¹⁸O substitution, appears at a different frequency than a band at 3626 cm⁻¹ previously assigned to a water molecule in GPR. However, the substitution of Gln105 for Leu105 in BPR leads to the appearance of both bands at 3642 and 3626 cm⁻¹, indicating the waters assigned in BPR and GPR exist in separate distinct locations and can coexist in the GPR-like Q105L mutant of BPR. These results indicate that there exist significant differences in the conformational changes occurring in these two types proteorhodopsin during the initial photoreaction despite their similar chromophore structures, which might reflect a different arrangement of water in the active site as well as substitution of a hydrophilic for hydrophobic residue at residue 105.

The lifetime of the asymmetric fundamental stretching 2218 cm^-1vibration of the anesthetic gas nitrous oxide ( N₂O ) dissolved in octanol and olive oil is reported. These solvents are model systems commonly used to assess anesthetic potency. Picosecond time-scale molecular dynamics simulations have suggested that protein dynamics or membrane dynamics play a role in the molecular mechanism of anesthetic action. Ultrafast infrared spectroscopy with 100 fs time resolution is an ideal tool to probe dynamics of anesthetic molecules on such timescales. Pump-probe studies centered at the peak of the vibrational band yield a lifetime of 55 ± 1 ps in olive oil and 52 ± 1 ps in octanol. The similarity of lifetimes suggests that energy relaxation of the anesthetic is determined primarily by the hydrophobic nature of the environment, consistent with models of anesthetic action. The results show that nitrous oxide is a good model system for probing anesthetic-solvent interactions using nonlinear infrared spectroscopy.