Structural studies on transmembrane proteins. 1. Model study using bacteriorhodopsin mutants containing single cysteine residues.
In developing new approaches to structural studies of polytopic transmembrane proteins, we have prepared bacteriorhodopsin mutants containing single cysteine residues at selected sites in different topological domains. Four such mutants were prepared: Gly-72----Cys and Ser-169----Cys in the presumed looped-out regions on the opposite sides of the membrane bilayer and Thr-90----Cys and Leu-92----Cys in the membrane-embedded helix C. The four mutants folded and regenerated the characteristic chromophore in detergent/phospholipid micelles and pumped protons like the wild-type bacteriorhodopsin. After reconstitution in asolectin vesicles, the sulfhydryl groups in the mutants Gly-72----Cys and Ser-169----Cys reacted with iodo[2-3H]acetic acid, while the sulfhydryl groups in the membrane-embedded mutants, Thr-90----Cys and Leu-92----Cys, did not. The sulfhydryl groups in all four mutants could be derivatized in the denatured state by reaction with iodoacetic acid or 6-acryloyl-2-(dimethylamino)naphthalene. Of these derivatives, the two from the mutants Gly-72----Cys and Ser-169----Cys folded like the wild-type bacterioopsin, whereas of the two from the helix C mutants, Thr-90----Cys and Leu-92----Cys, only the latter folded normally. However, the folding of Leu-92----Cys was also impaired when treated with the bulky 5-(iodoacetamido)fluorescein. The reactivity and the folding behavior of the cysteine mutants can thus report on the topographic domain as well as on the orientation of the helices within the membrane.
Flitsch
S L
SL
Department of Biology, Massachusetts Institute of Technology, Cambridge 02139.
Khorana
H G
HG
D001426:Bacterial Proteins / Q000096:biosynthesis / Q000235:genetics*; D001483:Base Sequence; D002851:Chromatography, High Pressure Liquid; D003545:Cysteine / Q000235:genetics*; D004247:DNA / Q000235:genetics; D004926:Escherichia coli / Q000032:analysis; D007461:Iodoacetates; D019807:Iodoacetic Acid; D008565:Membrane Proteins / Q000096:biosynthesis / Q000235:genetics*; D008956:Models, Chemical; D008969:Molecular Sequence Data; D009154:Mutation; D010957:Plasmids; D011487:Protein Conformation; D013438:Sulfhydryl Compounds
D001426:Bacterial Proteins; D007461:Iodoacetates; D008565:Membrane Proteins; D013438:Sulfhydryl Compounds; D004247:DNA; D003545:Cysteine; D019807:Iodoacetic Acid