Abstract
The previously described chymotryptic fragment of bacteriorhodopsin, C-2 (amino acids 1-71), is cleaved by 70% formic acid to two fragments, A-1 (amino acids 1-36) and A-2 (amino acids 37-71), which have been separated by high pressure liquid chromatography. The fragments A-1 and A-2, separately or together, are not able to replace C-2 in forming a stable bacteriorhodopsin-like complex with the fragment C-1 (amino acids 72-248) and all-trans-retinal. A second set of bacteriorhodopsin fragments, B-1 (amino acids 1-155) and B-2 (amino acids 156-248), have been prepared by sodium borohydride cleavage of bacteriorhodopsin. Following denaturation, fragments B-1 and B-2 reassociate in the presence of retinal to regenerate the native bacteriorhodopsin chromophore (approximately 40%). Fragment B-1 also interacts with fragment C-1 and all-trans-retinal to form a complex with spectral properties and secondary structure similar to those of bacteriorhodopsin. Vesicles prepared from the reconstituted fragment complexes (B-1 + B-2) or (B-1 + C-1), show proton pumping activities comparable to the previously described activity fragments C-1 and C-2.
Highlights
The previously described chymotryptic fragmentof aspects of BR structure and function
More, modifications or labeling of specific amino acids in chosen fragments provides a general approach for studying BR structure and function
We have used fragment reconstitution to confirm our earlier conclusion regarding the site of attachment of retinal within the protein [15, 16]
Summary
Cleauage of the Fragment C-2 by Formic Acid-The preparation of the fragment C-2 has previously been described [14]. Assay for Proton Pumping Activity-The procedures for preparation of reconstituted vesicles from BR or from fragments have been described [13],except that theinitial lipid/detergent mixture contains soybean phospholipids/CHAPS or H. hnlobium phospholipid/. Proton pumping previous results have shown that fragments C-1 and C-2 can activity of reconstituted vesicles was assayed as described previously reassociate and bind retinal to regenerate the native BR [13]. Cleavage of BR to Fragments B-1 and B-2 by NaBH, (PH performed on an unmodified Beckman 89OC sequencer as previously 1O)"When purple membrane was incubated with NaBH4(pH described [21] and a solid phase sequencer (sequemat Mini 15)by the procedure of Takagaki et al [22].
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