Structure of the N Intermediate of Bacteriorhodopsin Revealed by X-Ray Diffraction

• Published in: Proceedings of the National Academy of Sciences - PNAS Vol. 93; no. 4; pp. 1386 - 1390
• Main Authors: Kamikubo, Hironari, Kataoka, Mikio, Váró, György, Oka, Toshihiko, Tokunaga, Fumio, Needleman, Richard, Lanyi, Janos K.
• Format: Journal Article
• Language: English
• Published: United States National Academy of Sciences of the United States of America 20.02.1996; National Acad Sciences; National Academy of Sciences
• Subjects: Aspartic Acid - chemistry; Bacteria; Bacteriorhodopsins; Bacteriorhodopsins - chemistry; Bacteriorhodopsins - genetics; Bacteriorhodopsins - radiation effects; Biochemistry; Biophysics; Biotechnology; Correlation coefficients; Electron diffraction; Fourier Analysis; Halobacterium - chemistry; Halobacterium - genetics; Membrane Proteins - chemistry; Membrane Proteins - genetics; Membrane Proteins - radiation effects; Mutagenesis, Site-Directed; Photochemistry; Photons; Protein Conformation - radiation effects; Protons; Recombinant Proteins - chemistry; Recombinant Proteins - radiation effects; Schiff bases; Schiff Bases - chemistry; Schiff Bases - radiation effects; Wave diffraction; X ray diffraction; X-rays
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.93.4.1386

X-ray diffraction experiments revealed the structure of the N photointermediate of bacteriorhodopsin. Since the retinal Schiff base is reprotonated from Asp-96 during the M to N transition in the photocycle, and Asp-96 is reprotonated during the lifetime of the N intermediate, or immediately after, N is a key intermediate for understanding the light-driven proton pump. The N intermediate accumulates in large amounts during continuous illumination of the F171C mutant at pH 7 and 5 degrees C. Small but significant changes of the structure were detected in the x-ray diffraction profile under these conditions. The changes were reversible and reproducible. The difference Fourier map indicates that the major change occurs near helix F. The observed diffraction changes between N and the original state were essentially identical to the diffraction changes reported for the M intermediate of the D96N mutant of bacteriorhodopsin. Thus, we find that the protein conformations of the M and N intermediates of the photocyte are essentially the same, in spite of the fact that in M the Schiff base is unprotonated and in N it is protonated. The observed structural change near helix F will increase access of the Schiff base and Asp-96 to the cytoplasmic surface and facilitate the proton transfer events that begin with the decay of the M state.