Mark S. Braiman

Support planar germanium waveguides for infrared evanescent-wave sensing

We have fabricated miniature planar IR waveguides with thicknesses of 30-50 mum, consisting of 12-mm long, 2-mm wide strips of Ge supported on ZnS substrates. Evidence for efficient propagation of broadband IR light through these waveguides is provided by the presence of characteristic high- and low-frequency optical cutoffs of Ge; by the observation of an oscillatory interference pattern in the transmittance spectrum, which exhibits a dependence on waveguide thickness and propagation angle that closely matches waveguide theory; and by the detection of strong evanescent-wave absorption from small (2 mm(2)) droplets of liquid, e.g., water, on the waveguide surface. As also predicted by theory, the surface sensitivity (detected light absorbance per unit area of sample-waveguide contact) is shown to increase as a function of incidence or bevel angle.

KINETIC ANALYSIS OF TIME-RESOLVED INFRARED DIFFERENCE SPECTRA OF THE L and M INTERMEDIATES OF BACTERIORHODOPSIN*

Abstract– Infrared difference spectra with 4 cm−1 spectral resolution and 10‐u.s temporal resolution, obtained previously with a stroboscopic Fourier‐transform difference technique (Braiman et al., 1991, Proc. Natl. Acad. Sci. USA 88, 2388), were analyzed by means of a global exponential fitting procedure based on singular value decomposition. Using a simple linear kinetic model K → L → M for the bacteriorhodopsin (bR) photocycle in the time range10–1000 μ.s at 16.5°C, it was possible to generate bR → L and bR → M difference spectra with signal/noise ratios comparable to those obtainable with low temperature difference spectroscopy. The resulting time‐resolved bR → L and bR‐→ M difference spectra are both very similar to the corresponding static FTIR difference spectra obtained at 175 K and 250 K, respectively. In the bR → L spectrum, however, there are interesting differences that may indicate a greater degree of deprotonation of Asp‐96 when L is formed at physiological temperatures than when it is observed in a low‐temperature steady state.

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

BackgroundProteorhodopsin (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.ResultsA 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.ConclusionsThe 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).

A large photolysis-induced pKa increase of the chromophore counterion in bacteriorhodopsin: implications for ion transport mechanisms of retinal proteins.

The proton-pumping mechanism of bacteriorhodopsin is dependent on a photolysis-induced transfer of a proton from the retinylidene Schiff base chromophore to the aspartate-85 counterion. Up until now, this transfer was ascribed to a > 7-unit decrease in the pKa of the protonated Schiff base caused by photoisomerization of the retinal. However, a comparably large increase in the pKa of the Asp-85 acceptor also plays a role, as we show here with infrared measurements. Furthermore, the shifted vibrational frequency of the Asp-85 COOH group indicates a transient drop in the effective dielectric constant around Asp-85 to approximately 2 in the M photointermediate. This dielectric decrease would cause a > 40 kJ-mol-1 increase in free energy of the anionic form of Asp-85, fully explaining the observed pK alpha increase. An analogous photolysis-induced destabilization of the Schiff base counterion could initiate anion transport in the related protein, halorhodopsin, in which aspartate-85 is replaced by Cl- and the Schiff base proton is consequently never transferred.

Primary photochemistry of bacteriorhodopsin: comparison of Fourier transform infrared difference spectra with resonance Raman spectra.

Abstract —Fourier transform infrared (FTIR) difference spectra of the BR→rK transition in bacteriorhodopsin at 77→K are compared with analogous resonance Raman difference spectra obtained using a spinning sample cell at 77→K. The vibrational frequencies observed in the FTIR spectra of native purple membrane and of purple membrane regenerated with 15‐deuterioretinal are in good agreement with the frequencies observed in the Raman spectra, indicating that the lines in the FTIR difference spectra arise predominantly from retinal chromophore vibrations. This agreement confirms that the spinning cell method for obtaining resonance Raman spectra of K minimizes potential contributions from unwanted photoproducts. The unexpected similarity between the resonance Raman scattering intensities and the FTIR absorption intensities for BR and K is discussed in terms of the delocalized electronic structure of the chromophore. Finally, comparison of the Schiff base regions of the K Raman and FTIR spectra provide additional information on the assignment of its Schiff base vibration.

Structural comparison of metarhodopsin II, metarhodopsin III, and opsin based on kinetic analysis of Fourier transform infrared difference spectra

Fourier transform infrared difference spectra were measured at 30-s intervals after a complete bleach of rhodopsin (rho) samples at 20 degrees C and three different pH values. At each pH, all of the spectra could be fit globally to two exponential decay processes. Using a branched unimolecular kinetic model in which metarhodopsin II (meta II) is hydrolyzed to opsin and retinal both directly and through metarhodopsin III (meta III), we calculated rho-->meta II, rho-->meta III, and rho-->opsin difference spectra at each of the pH values and obtained estimates for the microscopic rate constants at each pH. Because of assumptions that had to be made about the branching ratio between the meta II decay pathways, some uncertainties remain in our calculated rho-->meta III difference spectrum at each pH. Nevertheless, our data covering long time ranges, especially those obtained at pH 8, place significant new constraints on the spectrum of meta III and thus on its structure. The rho-->meta II spectrum shows no significant pH dependence over the range examined (pH 5.5-8). However, the rho-->meta III and rho-->opsin spectra each include a limited subset of pH-dependent peaks, which are mostly attributable to titratable amino acid side chains. Our observations can be used to refine an earlier conclusion that the visual pigment refolds to a rhodopsin-like conformation during meta II decay (Rothschild, K.J., J. Gillespie, and W.J. DeGrip. 1987 Biophys. J. 51:345-350). Most of this refolding occurs in the same way at pH values ranging from 5.5 to 8 and whether meta II decays to meta III or opsin. Meta II displays unique spectral perturbations that are mostly attributable to a few residues, probably including three to four aspartic or glutamic acids and an arginine.

Nano- and Microsecond Time-Resolved FTIR Spectroscopy of the Halorhodopsin Photocycle

Abstract— Step‐scan Fourier transform infrared spectroscopy with 50 ns time resolution was applied to the early stages of the photocycle of halorhodopsin (hR) for the temperature range 3‐42° C. Kinetic data analysis with global fitting revealed two distinct kinetic processes associated with relaxations of the early red‐shifted photoproduct hK; these processes have time constants T1⋍ 280 ns and T2⋍ 360 μs at 20°C. Spectral features demonstrate that the T1 process corresponds to a transition between two distinct bathointermediates, hKE and hKL. The vibrational difference bands associated with both T1 and T2 transitions are spread throughout the whole 1800‐900 cm−1 range. However, the largest bands correspond to ethylenic C=C stretches, fingerprint C‐C stretches and hydrogen out‐of‐plane (HOOP) wags of the retinal chromophore. The time evolution of these difference bands indicate that both the T1 and T2 decay processes involve principally a relaxation of the chromophore and its immediate environment. The decay of the intense HOOP vibrations is nearly equally divided between the T1 and T2 processes, indicating a complex chromophore relaxation from a twisted nonrelaxed conformation in the primary (hKE) bathointermediate, to a less‐twisted structure in hKL, and finally to a roughly planar structure in the hypsochromically shifted hL intermediate. This conclusion is also supported by the unexpectedly large positive entropy of activation observed for the T1 process. The two relaxations from hKE to hL are largely analogous to corresponding relaxations (KE→ KL→ L) in the bacterior‐hodopsin photocycle, except that the second step is slowed down by over 200‐fold in hR.

Expression and characterization of the fourth repeat of Xenopus interphotoreceptor retinoid-binding protein in E. coli

Interphotoreceptor retinoid-binding protein (IRBP) is an extracellular glycolipoprotein which in higher vertebrates has a 4-repeat structure and carries endogenous vitamin A and fatty acids. The location of IRBPs 1-2 binding sites for retinol is unknown. To begin to understand which repeat(s) are responsible for ligand-binding, we expressed the fourth repeat of Xenopus IRBP in E. coli to determine if it could by itself bind all-trans retinol. Our expression studies used a polyhistidine fusion domain to purify the recombinant protein directly from inclusion bodies. The fusion protein could be renatured without aggregation if refolded at a sufficiently dilute concentration (< 3 microM). The recombinant fourth repeat of Xenopus IRBP binds [3H]all-trans retinol and the fluorescence of this ligand increases 8-fold upon binding. The binding is saturable with a Kd = 0.4 microM. The expression of recombinant IRBP fragments as fusion proteins in prokaryotes will be useful for defining the structural requirements for ligand binding by this interesting protein.

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

Time-resolved Fourier transform infrared (FTIR) difference spectra of the halorhodopsin (hR) photocycle have been collected from 3 micros to 100 ms in saturating concentrations of KCl or KBr. Kinetic analysis of these data revealed two decay processes, with time constants of tau(1) approximately 150 micros and tau(2) approximately 16 ms in the presence of either halide, with tau(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 tau(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-, the hL features are clearly more dominant than in Cl-. Therefore, the state present before tau(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- produces a much more significant back-reaction from hL(1) to hK(L) than does Br-. The halide dependence of this back-reaction could therefore explain the halide selectivity of the halorhodopsin anion pump.

Vibrational spectra of individual millimeter-size membrane patches using miniature infrared waveguides

We have used miniature planar IR waveguides, consisting of Ge strips 30-50 microm thick and 2 mm wide, as evanescent-wave sensors to detect the mid-(IR) evanescent-wave absorbance spectra of small areas of biomolecular monolayers and multilayers. Examples include picomolar quantities of an integral transmembrane protein (bacteriorhodopsin) and lipid (dimyristoyl phosphatidylcholine). IR bands due to the protein and lipid components of the plasma membrane of individual 1.5-mm-diameter devitellinized Xenopus laevis oocytes, submerged in buffer and sticking to the waveguide surface, were also detected. A significant improvement in sensitivity was observed, as compared to previous sizes and geometries of evanescent-wave sensors (e.g., commercially available internal reflection elements or tapered optical fibers). These measurements suggest the feasibility of using such miniature supported planar IR waveguides to observe structural changes in transmembrane proteins functioning in vivo in single cells.