A Three-Dimensional Difference Map of the N Intermediate in the Bacteriorhodopsin Photocycle: Part of the F Helix Tilts in the M to N Transition

Abstract

The N intermediate of the bacteriorhodopsin photocycle was trapped for electron diffraction studies in glucose-embedded specimens of the site-directed mutant Phe219 --> Leu. At neutral pH, the N-bR difference Fourier transform infrared spectrum of this mutant is indistinguishable from published difference spectra obtained for wild-type bacteriorhodopsin at alkaline pH. An electron diffraction difference map of the N intermediate in projection shows large differences near the F and the G helix, which are very similar to the features seen in the M intermediates of the Asp96 --> Gly mutant [Subramaniam et al. (1993) EMBO J. 12, 1-8]. This similarity was anticipated on the basis of Fourier transform infrared data, which have shown that the M intermediate trapped in Asp96 mutants already has the protein structure of the N intermediate [Sasaki et al. (1992) J. Biol. Chem. 267, 20782-20786]. A preliminary three-dimensional difference map of the N intermediate, calculated from electron diffraction data of samples tilted at 25 degrees, clearly shows that the change on the F helix consists of an outward movement of the cytoplasmic end of the helix. In addition, the cytoplasmic side of the G helix moves or becomes more ordered. Comparison with published difference maps of the M intermediate indicates that the F helix tilt occurs in the M to N transition, but the G helix change represents an earlier step in the photocycle.