Chemical reconstitution of a chloride pump inactivated by a single point mutation.
The arginine residue R108 plays an essential role in the transport mechanism of the light-driven anion pump halorhodopsin (HR) as demonstrated by complete inactivation of chloride transport in mutant HR-R108Q. In the presence of substrate anions, guanidinium ions bind to the mutant protein with affinities in the mM range, thereby restoring transport activity and photochemical properties of wild type. One guanidinium ion and one anion are bound per molecule of HR-R108Q. For HR wild type, HR-R108Q-guanidinium and HR-R108K, differences in transport activity and anion selectivity are found which may be explained by effects of anion solvation. The agreement between light-induced FTIR difference spectra of HR wild type and HR-R108Q-guanidinium demonstrates that no structural changes occur in the reconstituted mutant and that the photoreactions of wild type and reconstituted mutant are identical. Furthermore, an IR absorbance band of the guanidino group of R108 can be identified at 1695/1688 cm-1. In HR-R108Q, a guanidinium ion binding close to the mutated residue is proposed to mimick the role of the R108 side chain as the anion uptake site. Thus the wild type reaction mechanism is reconstituted.
Rüdiger
M
M
Max-Planck-Institut für Biochemie, Martinsried, Germany.
Haupts
U
U
Gerwert
K
K
Oesterhelt
D
D
D000838:Anions / Q000378:metabolism; D001436:Bacteriorhodopsins / Q000187:drug effects / Q000235:genetics / Q000378:metabolism* / Q000528:radiation effects; D001692:Biological Transport / Q000235:genetics / Q000502:physiology; D002712:Chlorides / Q000378:metabolism*; D019791:Guanidine; D006146:Guanidines / Q000378:metabolism / Q000494:pharmacology; D006217:Halobacterium / Q000187:drug effects / Q000235:genetics / Q000378:metabolism* / Q000528:radiation effects; D025602:Halorhodopsins; D016623:Ion Pumps / Q000187:drug effects / Q000235:genetics / Q000378:metabolism* / Q000528:radiation effects; D010782:Photolysis; D017354:Point Mutation; D013053:Spectrophotometry; D017550:Spectroscopy, Fourier Transform Infrared
D000838:Anions; D002712:Chlorides; D006146:Guanidines; D025602:Halorhodopsins; D016623:Ion Pumps; D001436:Bacteriorhodopsins; D019791:Guanidine