Bacteriorhodopsin site-directed mutagenesis

Energy landscape of bacteriorhodopsin from AFM studies has been reviewed in this review. Recently we have embarked on a project to determine the unfolding pathway of bR mutants where mutations in F and G helices were introduced by site-directed mutagenesis,e.g. [E9Q/E194Q/E204Q] bR. This mutant was designed... 

Bacteriorhodopsin translocates one H ion per photon which causes the aW-trans — 13-c 5 photoisomerization of chromophore, retinal. At least two H -acceptor groups are shown to be directly involved in the H transfer by bacteriorhodopsin, namely, (a) the Schiff base forming a link between the retinal and the e-amino group of Lys-216, and (b) the Asp-96 carboxylic group. The involvement of the Schiff base is confirmed by many independent pieces of evidence (e.g., the electrogenic H transfer disappears at a pH below 3.5, i.e., below the pK value of the Schiff base in the M-intermediate of bacteriorhodopsin photocycle reviewed in ref. [7]). As to Asp-96, its participation in the H transfer relay was recently demonstrated by site-directed mutagenesis studies [13-19]. 

The experimental structure of bR determined at atomic resolution from cryoelectron microscopy and X-ray crystallography revealed a channel containing the Schiff base of the retinal chromophore (27, 28). Site-directed mutagenesis and vibrational spectroscopy experiments have enabled the identification of polar residues in the channel involved in the proton transfer pathway (29-32). Recent work on bacteriorhodopsin has concentrated on hydration and conformational thermodynamics. 

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. 

Biomaterials are seldom suitable for device construction, but with the advances in synthetic chemistry and in genetic engineering (site-directed mutagenesis), raw biomaterials can be modified to suit the technological needs. Owing to its extraordinary stability, bacteriorhodopsin was one of the first biomaterials to be successfully utilized for device construction. Vsevolodov... 

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. 

Finally, protein bands can edso be identified by site-directed mutagenesis. Again, the method has been applied to bacteriorhodopsin, and a large... 

---