Anhydrous Monoalkylguanidines in Aprotic and Nonpolar Solvents: Models for Deprotonated Arginine Side Chains in Membrane Environments.

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.

Experimental and Computational Modeling of H-Bonded Arginine-Tyrosine Groupings in Aprotic Environments.

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.

Computations on the primary photoreaction of Br2 with CO2: stepwise vs concerted addition of Br atoms.

It was proposed previously that Br2-sensitized photolysis of liquid CO2 proceeds through a metastable primary photoproduct, CO2Br2. Possible mechanisms for such a photoreaction are explored here computationally. First, it is shown that the CO2Br radical is not stable in any geometry. This rules out a free-radical mechanism, for example, photochemical splitting of Br2 followed by stepwise addition of Br atoms to CO2-which in turn accounts for the lack of previously observed Br2+CO2 photochemistry in gas phases. A possible alternative mechanism in liquid phase is formation of a weakly bound CO2:Br2 complex, followed by concerted photoaddition of Br2. This hypothesis is suggested by the previously published spectroscopic detection of a binary CO2:Br2 complex in the supersonically cooled gas phase. We compute a global binding-energy minimum of -6.2 kJ mol(-1) for such complexes, in a linear geometry. Two additional local minima were computed for perpendicular (C2v) and nearly parallel asymmetric planar geometries, both with binding energies near -5.4 kJ mol(-1). In these two latter geometries, C-Br and O-Br bond distances are simultaneously in the range of 3.5-3.8 Å, that is, perhaps suitable for a concerted photoaddition under the temperature and pressure conditions where Br2 + CO2 photochemistry has been observed.

Bromine-sensitized solar photolysis of CO2.

Direct photochemical reduction of CO(2) has generally been accomplished by using transition-metal compounds as electron transfer reagents. Here, we show that elemental bromine can function as an alternative photosensitizer. When sunlight is tightly focused on mixtures of CO(2) and Br(2), in the presence of a polar adsorbent such as silica gel, glass wool, alumina, or titania, a metastable red adduct is formed within seconds and concentrates at the point of illumination. Further illumination causes deposition of a stable black film on the polar adsorbent. Mass spectrometry of the cold-trapped red intermediate shows clusters of peaks corresponding to the expected distribution of isotopomers of C(2)O(4)Br(4)(+), as well as of C(2)O(4)Br(3)(+). DFT computations indicate that the lowest-energy species with the formula C(2)O(4)Br(4) is trans-2,4-dibromo-2,4-dihypobromo-1,3-dioxetane. Formation of this molecule from (2CO(2) + 2Br(2)) would require a minimum of 3 visible photons, two of which would hypothetically be used in formation of as-yet undetected CO(2)Br(2) and the third, in a subsequent photodimerization. By elemental analysis, the final amorphous solid product contains a C/Br atomic ratio >12, suggesting that Br(2) is acting photocatalytically. Even with a poorly optimized optical system, the reaction rate has reached as high as 1.6 mg reduced C with 40 s of solar collection using a 30 cm diameter paraboloid reflector. This rate is consistent with the storage of approximately 1% of incident solar energy.

Infrared and visible absolute and difference spectra of bacteriorhodopsin photocycle intermediates.

We have used new kinetic fitting procedures to obtain infrared (IR) absolute spectra for intermediates of the main bacteriorhodopsin (bR) photocycle(s). The linear-algebra-based procedures of Hendler et al. (J. Phys. Chem. B, 105, 3319-3228 (2001)) for obtaining clean absolute visible spectra of bR photocycle intermediates were adapted for use with IR data. This led to isolation, for the first time, of corresponding clean absolute IR spectra, including the separation of the M intermediate into its M(F) and M(S) components from parallel photocycles. This in turn permitted the computation of clean IR difference spectra between pairs of successive intermediates, allowing for the most rigorous analysis to date of changes occurring at each step of the photocycle. The statistical accuracy of the spectral calculation methods allows us to identify, with great confidence, new spectral features. One of these is a very strong differential IR band at 1650 cm(-1) for the L intermediate at room temperature that is not present in analogous L spectra measured at cryogenic temperatures. This band, in one of the noisiest spectral regions, has not been identified in any previous time-resolved IR papers, although retrospectively it is apparent as one of the strongest L absorbance changes in their raw data, considered collectively. Additionally, our results are most consistent with Arg82 as the primary proton-release group (PRG), rather than a protonated water cluster or H-bonded grouping of carboxylic residues. Notably, the Arg82 deprotonation occurs exclusively in the M(F) pathway of the parallel cycles model of the photocycle.

Weakened coupling of conserved arginine to the proteorhodopsin chromophore and its counterion implies structural differences from bacteriorhodopsin.

In wild-type proteorhodopsin (pR), titration of the chromophore's counterion Asp(97) occurs with a pK(a) of 8.2+/-0.1. R94C mutation reduces this slightly to 7.0+/-0.2, irrespective of treatment with ethylguanidinium. This contrasts with the homologous archaeal protein bacteriorhodopsin (bR), where R82C mutation was previously shown to elevate the pK(a) of Asp(85) by approximately 5 units, while reconstitution with ethylguanidinium restores it nearly to the wild-type value of 2.5. We conclude there is much weaker electrostatic coupling between Arg(94) and Asp(97) in the unphotolyzed state of pR, in comparison to Arg(82) and Asp(85) in bR. Therefore, while fast light-driven H(+) release may depend on these two residues in pR as in bR, no tightly conserved pre-photolysis configuration of them is required.

Time-resolved FTIR spectroscopy of the photointermediates involved in fast transient H+ release by proteorhodopsin.

Proteorhodopsin (pR) is a homologue of bacteriorhodopsin (bR) that has been recently discovered in oceanic bacterioplankton. Like bR, pR functions as a light-driven proton pump. As previously characterized by laser flash induced absorption spectroscopy (Krebs, R. A.; Alexiev, U.; Partha, R.; DeVita, A. M.; Braiman, M. S. BMC Physiol. 2002, 2, 5), the pR photocycle shows evidence of light-induced H(+) release on the 10-50 micros time scale, and of substantial accumulation of the M intermediate, only at pH values above 9 and after reconstitution into phospholipid followed by extensive washing to remove detergent. We have therefore measured the time-resolved FTIR difference spectra of pR intermediates reconstituted into DMPC vesicles at pH 9.5. A mixture of K- and L-like intermediates, characterized by a 1516 cm(-1) positive band and a 1742 cm(-1) negative band respectively, appears within 20 micros after photolysis. This mixture decays to an M-like state, with a clear band at 1756 cm(-1) due to protonation of Asp-97. The 50-70 micros rise of M at pH 9.5 is similar to (but a little slower than) the rise times for M formation and H(+) release that were reported earlier based on flash photolysis measurements of pR reconstituted into phospholipids with shorter acyl chains. We conclude that, at pH 9.5, H(+) release occurs while Asp-97 is still protonated; i.e., this aspartic acid cannot be the H(+) release group observed by flash photolysis under similar conditions.

Modeling amino acid side chains in proteins: 15N NMR spectra of guanidino groups in nonpolar environments.

Natural-abundance 15N NMR spectroscopy on dodecylguanidine reveals solvent and protonation effects that model those that could occur for the arginine side chain in proteins. Our results demonstrate that the 15N chemical shifts of the terminal guanine nitrogens strongly depend on the solvent chosen for measurements. A polar H-bond-donating solvent like water has strongly deshielding effects on the neutral guanidine group (with the latter acting predominantly as an H-bond acceptor). As a result, a substantial upfield shift occurs when neutral guanidine is dissolved instead in a non-H-bonding solvent (chloroform). These solvent effects can be as large as those induced by protonation changes. This limits the ability of 15N chemical shifts to distinguish the protonation state of the arginine side chain, at least without specific knowledge of its environment. These results help to reconcile previous interpretations about the protonation state arg-82 in the M state of bacteriorhodopsin based on FTIR and 15N NMR spectroscopy. That is, contrary to earlier conclusions from solid-state NMR, the side chain of arg-82 could undergo a deprotonation between the bR and M states, but only if it also experienced a significant decrease in the H-bonding character and polarity of its environment. In fact, the average 15N chemical shift of the two Neta of arg-82 in bacteriorhodopsin's M intermediate (from the previous NMR measurements) is 17 ppm upfield from the corresponding value for the deprotonated arginine side chain in aqueous solution at pH >14, but only 3 ppm upfield from the value for deprotonated dodecylguanidine in chloroform.

Role of arginine-82 in fast proton release during the bacteriorhodopsin photocycle: a time-resolved FT-IR study of purple membranes containing 15N-labeled arginine.

Arginine-82 has long been recognized as an important residue in bacteriorhodopsin (bR), because its mutation usually results in loss of fast H(+) release, an important step in the normal light-induced H(+) transport mechanism. To help to clarify the structural changes in Arg-82 associated with the H(+)-release step, we have measured time-resolved FT-IR difference spectra of wild-type bR containing either natural-abundance isotopes ((14)N-Arg-bR) or all seven arginines selectively and uniformly labeled with (15)N at the two eta-nitrogens ((15)N-Arg-bR). Comparison of the spectra from the two isotopic variants shows that a 1556 cm(-1) vibrational difference band due to the M photocycle intermediate of (14)N-Arg-bR loses substantial intensity in (15)N-Arg-bR. However, this isotope-sensitive arginine vibrational difference band is only observed at pH 7 and not at pH 4 where fast H(+) release is blocked. These observations support the earlier conclusion, based on site-directed mutagenesis and chemical labeling, that a strong C-N stretch vibration of Arg-82 can be assigned to a highly perturbed frequency near 1555 cm(-1) in the M state of wild-type bR [Hutson et al. (2000) Biochemistry 39, 13189-13200]. Furthermore, alkylguanidine model compound spectra indicate that the unusually low arginine C-N stretch frequency in the M state is consistent with a nearly stoichiometric light-induced deprotonation of an arginine side chain within bR, presumably arginine-82.

Characterization of supported cylinder-planar germanium waveguide sensors with synchrotron infrared radiation.

Cylinder-planar Ge waveguides are being developed as evanescent-wave sensors for chemical microanalysis. The only non-planar surface is a cylinder section having a 300-mm radius of curvature. This confers a symmetric taper, allowing for direct coupling into and out of the waveguide's 1-mm(2) end faces while obtaining multiple reflections at the central <30-microm-thick sensing region. Ray-optic calculations indicate that the propagation angle at the central minimum has a strong nonlinear dependence on both angle and vertical position of the input ray. This results in rather inefficient coupling of input light into the off-axis modes that are most useful for evanescent-wave absorption spectroscopy. Mode-specific performance of the cylinder-planar waveguides has also been investigated experimentally. As compared to a blackbody source, the much greater brightness of synchrotron-generated infrared (IR) radiation allows a similar total energy throughput, but restricted to a smaller fraction of the allowed waveguide modes. However, such angle-selective excitation results in a strong oscillatory interference pattern in the transmission spectra. These spectral oscillations are the principal technical limitation on using synchrotron radiation to measure evanescent-wave absorption spectra with the thin waveguides.

Detection of fast light-activated H+ release and M intermediate formation from proteorhodopsin.

BACKGROUND: Proteorhodopsin (pR) is a light-activated proton pump homologous to bacteriorhodopsin and recently discovered in oceanic gamma-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. RESULTS: 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 micros) as reported for monomeric bR in detergent. CONCLUSIONS: 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).