Application of Doubled-Angle Phase Correction Method to Time-Resolved Step-Scan FT-IR Spectra

Abstract Ž . To increase the sensitivity with which time-resolved Fourier transform infrared FT-IR difference spectra are measured, the detector is often AC-coupled. Thus, the measured interferograms correspond to spectra with both positive and negative intensities. The presence of signed intensities presents problems for the standard Mertz and Forman phase correction methods. The Mertz Signed phase correction method was designed to handle signed intensities, but the smoothing inherent in calculating the phase angles at reduced resolution introduces other errors in AC-coupled spectra produced with this algorithm. These errors are evident as signal remaining along the imaginary axis after phase correction. A new approach to phase correction, the Doubled-Angle method, can directly correct the phases of transient AC-coupled spectra without the w Ž . x need for a DC interferogram M.S. Hutson, M.S. Braiman, Appl. Spectrosc. 52 1998 974 . When this method was applied to the transient AC interferograms measured after photolysis of bacteriorhodopsin, the signal was fully rotated onto the real axis following phase correction. Here, we show that the Doubled-Angle method can be applied to time-resolved difference FT-IR spectra of halorhodopsin, a more demanding biological system due to its intrinsically small differential absorption signals.

Arginine to Glutamine Substitutions in the Fourth Module of Xenopus Interphotoreceptor Retinoid-Binding Protein

Interphotoreceptor retinoid-binding protein (IRBP) is unusual for a lipid-binding protein in that its gene is expressed uniquely by cells of photoreceptor origin and consists of four homologous repeats, each coding for a module of~300 amino acid residues. All-trans retinol binding domains, which appear to be present in each module, are composed of conserved hydrophobic regions [Baer et al, Exp Eye Res 1998; 66:249-262]. Here we investigate the role of highly conserved arginines contained in these regions

Halide Dependence of the Halorhodopsin Photocycle as Measured by Time-Resolved Infrared Spectra

ABSTRACT Time-resolved Fourier transform infrared (FTIR) difference spectra of the halorhodopsin (hR) photocycle have been collected from 3 s to 100 ms in saturating concentrations of KCl or KBr. Kinetic analysis of these data revealed two decay processes, with time constants of 1 Ӎ 150 s and 2 Ӎ 16 ms in the presence of either halide, with 2 describing the return to the starting (hR) state. Comparison to previous low-temperature FTIR spectra of hR intermediates confirms that characteristic hK and hL spectral features are both present before the 1 decay, in a state previously defined as hK L (Dioumaev, A., and M. Braiman. 1997. Photochem. Photobiol. 66:755-763). However, the relative sizes of these features depend on which halide is present. In Br Ϫ , the hL features are clearly more dominant than in Cl Ϫ . Therefore, the state present before 1 is probably best described as an hK L /hL 1 equilibrium, instead of a single hK L state. Different halides affect the relative amounts of hK L and hL 1 present, i.e., Cl Ϫ produces a much more significant back-reaction from hL 1 to hK L than does Br Ϫ . The halide dependence of this back-reaction could therefore explain the halide selectivity of the halorhodopsin anion pump

Bromine-Sensitized Solar Photolysis of CO₂

Direct photochemical reduction of CO₂ 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 CO₂ and Br₂, 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 C₂O₄Br₄⁺, as well as of C₂O₄Br₃⁺. DFT computations indicate that the lowest-energy species with the formula C₂O₄Br₄ is trans-2,4-dibromo-2,4-dihypobromo-1,3-dioxetane. Formation of this molecule from (2CO₂ + 2Br₂) would require a minimum of 3 visible photons, two of which would hypothetically be used in formation of as-yet undetected CO₂Br₂ and the third, in a subsequent photodimerization. By elemental analysis, the final amorphous solid product contains a C/Br atomic ratio >12, suggesting that Br₂ 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

Symmetrically tapered <30-micron-thick quasi-planar Ge waveguides as chemical sensors for microanalysis

Symmetrically tapered planar IR waveguides have been fabricated by starting with a ZnS coated concave piece of single-crystal Ge, embedding it in an epoxide resin as a supporting substrate, and then grinding and polishing a planar surface until the thickness at the taper minimum is &lt;30 &mu;m. Such tapering is expected to enhance a waveguide\u27s sensitivity as an evanescent wave sensor by maximizing the amount of evanescent wave energy present at the thinnest part of the waveguide. As predicted by theory, the surface sensitivity, i.e., the absorbance signal per molecule in contact with the sensing region, increases with decreasing thickness of the tapered region even while the total energy throughput decreases. The signal-to-noise ratio obtained depends very strongly on the quality of the polished surfaces of the waveguides. The surface sensitivity is superior to that obtained with a commercial Ge attenuated total reflection (ATR) accessory for several types of sample, including thin films (&lt;10 ng) and small volumes (&lt;1 &mu;L) of volatile solvents. By using the waveguides, light-induced structural changes in the protein bacteriorhodopsin were observable using samples as small as &sim;50 pmol (&sim;1 &mu;g). In addition, the waveguide sensors can reveal the surface compositions on a single human hair, pointing to their promise as a tool for forensic fiber analysis

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 Δ<i>H</i>° = −74 ± 4 kJ mol^–1 is deduced for the formation of H-bonded <i>p</i>-cresol/dodecylguanidine in hexane. Δ<i>E</i> = −71 kJ mol^–1 is computed with density functional theory (in vacuo). In dimethyl sulfoxide or crystals, (<i>p</i>-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

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 Δ<i>H</i>° = −74 ± 4 kJ mol^–1 is deduced for the formation of H-bonded <i>p</i>-cresol/dodecylguanidine in hexane. Δ<i>E</i> = −71 kJ mol^–1 is computed with density functional theory (in vacuo). In dimethyl sulfoxide or crystals, (<i>p</i>-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