Proteorhodopsin (pR) is a light-activated proton pump homologous to bacteriorhodopsin and recently discovered in oceanic γ-proteobacteria. One perplexing difference between these two proteins is the absence in pR of homologues of bR residues Glu-194 and Glu-204. These two residues, along with Arg-82, have been implicated in light-activated fast H+ release to the extracellular medium in bR. It is therefore uncertain that pR carries out its physiological activity using a mechanism that is completely homologous to that of bR. A pR purification procedure is described that utilizes Phenylsepharose™ and hydroxylapatite columns and yields 85% (w/w) purity. Through SDS-PAGE of the pure protein, the molecular weight of E.-coli-produced pR was determined to be 36,000, approximately 9,000 more than the 27,000 predicted by the DNA sequence. Post-translational modification of one or more of the cysteine residues accounts for 5 kDa of the weight difference as measured on a cys-less pR mutant. At pH 9.5 and in the presence of octylglucoside and diheptanoylphosphotidylcholine, flash photolysis results in fast H+ release and a 400-nm absorbing (M-like) photoproduct. Both of these occur with a similar rise time (4–10 μs) as reported for monomeric bR in detergent. The presence of fast H+ release in pR indicates that either different groups are responsible for fast H+ release in pR and bR (i.e. that the H+ release group is not highly conserved); or, that the H+ release group is conserved and is therefore likely Arg-94 itself in pR (and Arg-82 in bR, correspondingly).

H-bonds between neutral tyrosine and arginine in nonpolar environments are modeled by small-molecule phenol/guanidine complexes. From the temperature and concentration dependence of UV spectra, a value of ΔH° = −74 ± 4 kJ mol^–1 is deduced for the formation of H-bonded p-cresol/dodecylguanidine in hexane. ΔE = −71 kJ mol^–1 is computed with density functional theory (in vacuo). In dimethyl sulfoxide or crystals, (p-phenolyl)alkylguanidines form head-to-tail homodimers with two strong H-bonding interactions, as evidenced by UV, IR, and NMR spectral shifts, strong IR continuum absorbance bands, and short O···N distances in X-ray crystal structures. Phenol/alkylguanidine H-bonded complexes consist of polarizable rapidly interconverting tautomers, with the proton shift from phenol to guanidine increasing with increase in the polarity of the aprotic solvent. As measured by NMR, both groups in these strongly H-bonded neutral complexes can simultaneously appear to be predominantly protonated. These systems serve as models for the hypothetical hydrogen-Bonded Uncharged (aRginine + tYrosine), or “BU(RY)”, motifs in membrane proteins.

We have used new kinetic fitting procedures to obtain infrared (IR) absolute spectra for intermediates of the main bacteriorhodopsin (bR) photocycle(s). The lin...

In this study, the synthesis of crystalline dodecylguanidine free base and its spectroscopic characterization in nonpolar environments are described. IR as well as ¹H and ¹⁵N NMR spectra of the free base dissolved in aprotic solvents are substantially different from the previously reported spectra of arginine, or other monoalkylguanidinium compounds, at high hydroxide concentrations. The current results provide improved modeling for the spectroscopic signals that would be expected from a deprotonated arginine in a nonpolar environment. On the basis of our spectra of the authentic dodecylguanidine free base, addition of large amounts of aqueous hydroxide to arginine or other monoalklyguanidinium salts does not deprotonate them. Instead, hydroxide addition leads to the formation of a guanidinium hydroxide complex, with a dissociation constant near ∼500 mM that accounts for the established arginine pK value of ∼13.7. We also report a method for synthesizing a compound containing both phenol and free-base guanidine groups, linked by a dodecyl chain that should be generalizable to other hydrocarbon linkers. Such alkyl-guanidine and phenolyl-alkyl-guanidine compounds can serve as small-molecule models for the conserved arginine–tyrosine groupings that have been observed in crystallographic structures of both microbial rhodopsins and G-protein-coupled receptors.