Halorhodopsin from Natronomonas pharaonis (pHR) functions as a light-driven halide ion pump. In the presence of halide ions, the photochemical reaction of pHR is described by the scheme: K→ L1 → L2 → N → O → pHR' → pHR. Here, we report light-induced structural changes of the pHR-bromide complex observed in the C2 crystal. In the L1-to-L2 transition, the bromide ion that initially exists in the extracellular vicinity of retinal moves across the retinal Schiff base. Upon the formation of the N state with a bromide ion bound to the cytoplasmic vicinity of the retinal Schiff base, the cytoplasmic half of helix F moves outward to create a water channel in the cytoplasmic interhelical space, whereas the extracellular half of helix C moves inward. During the transition from N to an N-like reaction state with retinal assuming the 13-cis/15-syn configuration, the translocated bromide ion is released into the cytoplasmic medium. Subsequently, helix F relaxes into its original conformation, generating the O state. Anion uptake from the extracellular side occurs when helix C relaxes into its original conformation. These structural data provide insight into the structural basis of unidirectional anion transport.
Halorhodopsin from Natronomonas pharaonis (pHR), a retinylidene protein that functions as a light-driven chloride ion pump, is converted into a proton pump in the presence of azide ion. To clarify this conversion, we investigated light-induced structural changes in pHR using a C2 crystal that was prepared in the presence of Cl(-) and subsequently soaked in a solution containing azide ion. When the pHR-azide complex was illuminated at pH 9, a profound outward movement (∼4 Å) of the cytoplasmic half of helix F was observed in a subunit with the EF loop facing an open space. This movement created a long water channel between the retinal Schiff base and the cytoplasmic surface, along which a proton could be transported. Meanwhile, the middle moiety of helix C moved inward, leading to shrinkage of the primary anion-binding site (site I), and the azide molecule in site I was expelled out to the extracellular medium. The results suggest that the cytoplasmic half of helix F and the middle moiety of helix C act as different types of valves for active proton transport.
SummaryMicro capillary columns were successfully applied to liquid chromatography by employing the principles used in micro high performance liquid chromatography. Fundamental investigations on the use of capillary columns in LC were performed for the various column parameters. Good separations of five aromatic hydrocarbons and four kinds of phthalic esters were obtained on a 62 pm I.D. capillary column, coated with SE-30.
In liquid chromatography, diffusion coefficients in the mobile phase (Dm) are quite small (-10-5-106 cm2/sec). The second term which is represented by 2DmlU can thus be ignored. The first term, which described the contribution by eddy diffusion, 2 A d, , is also negligible in open tubular columns (A is an eddy diffusion constant and d, is the particle diameter). Provided that the stationary phase consists of a uniform film on the glass surface, the third term can be written as follows, according to Giddings [I 91:where d is the depth (or thickness) of the stationary phase, Ds is the diffusion coefficient for the stationary phase, R is the retention ratio and u is the linear velocity of the mobile phase. If the mobile phase flow pattern is laminar in a round capillary tube, the fourth term can be written as:( 3) where rc is the column radius. Equation (1) can be written in the following final form for capillary columns in LC:Equation ( 2. H is inversely proportional to diffusion coefficients in both phases.3. H is related to the thickness of the stationary phase and the square of the capillary column radius. Hm becomes very large and low efficiency results. The column radius in capillary LC should be reduced to onehundredth, namely 2.5 pm, to get a contribution of Hm to H which is comparable to that in GC. If the linear velocity of capillary LC set at one-tenth of GC, the optimum column I. D. becomes about 8-16 pm.In addition, the diffusion coefficient for the stationary phase may also be considerably smaller in LC than in GC, as liquid chromatographic operations are usually performed at room temperature. Thus, it is preferable to make the thickness of the stationary phase as small as possible.
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