Mutant strain, bacteriorhodopsin

Halorhodopsiti. In addition to bacteriorhodopsin there are three other retinal-containing proteins in membranes of halobacteria. From mutant strains lacking bacteriorhodopsin the second protein, halorhodopsin, has been isolated. It acts as a light-driven chloride ion pump, transporting Cl from outside to inside. Potassium ions follow, and the pump provides a means for these bacteria to accumulate KC1 to balance the high external osmotic pressure of the environment in which they live.578 The amino acid sequences of halorhodopsins from several species are very similar to those of bacteriorhodopsin as is the three-dimensional structure.589 However, the important proton-carrying residues D85 and D96 of bacteriorhodopsin are replaced by threonine and alanine, respectively, in halorhodopsin.590 Halorhodopsin (hR)... 

Independent support for the validity of the foregoing component analysis is provided by experiments carried out with a mutant bacteriorhodopsin. Purple membranes were isolated from a mutant strain of Halobacterium halobium in which a point mutation at residue 212 (aspartic acid replaced by asparagine) was carried out by a new method of site-directed mutagenesis and expression (43, 44). The photosignal was found to be pH-independent in the range of pH 4-11 (45, 46). This photosignal was found to be a pure B1 component because its time course could be superimposed, after normalization, with that of the pure B1 component observed in a multilayered mutant bacteriorhodopsin film. Thus, Nature does indeed decompose the photosignal in accordance with the outlined component analysis. In other words, the B1 component as defined is indeed a natural entity. 

Site-directed mutagenesis as applied to bacteriorhodopsin was a difficult and labor-intensive procedure that used E. coli as the expression system (79, 80). Recently, Needleman s group successfully developed a new expression system that uses a bacteriorhodopsin-deficient mutant strain of H. halobium (43, 44). Preliminary results were quite encouraging. Unlike the mutants expressed in E. coli, the new method produces mutant bacteriorhodopsins with properties that differ from the protein expressed by E. coli. Presumably this difference occurs because correct folding into three-dimensional structures is more likely in the natural host than in its surrogate. Denaturation of the mutant proteins is further avoided because reconstitution is unnecessary. Our preliminary results show that the fast photoelectric signal can be drastically altered by a judiciously chosen point mutation. 

So far all stimulatory processes of the halobacterial cell depend on fumarate as seen by the fact that smooth swimming mutant cells of strain M415 do not respond to any stimulus. This also holds true for the step-down photophobic response mediated by bacteriorhodopsin[5]. This points to the role of a central metabolite as an integrative tool for measurement of the metabolic state of the cell. Especially fumarate would allow us to measure and compare activity states of electron transport, fermentation and photosynthesis (see Fig. 2). 

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