Tryptophan interactions in bacteriorhodopsin: a heteronuclear solid-state NMR study.
The bulky and amphiphilic nature of tryptophan residues makes them particularly interesting components of proteins. In bacteriorhodopsin, four of the eight tryptophan residues are in the active site, forming parts of the retinal binding pocket. In this work, we use solid-state NMR to study the interactions of the tryptophan residues in wild-type bacteriorhodopsin, in the resting state, and in critical intermediates of the proton-motive photocycle. The range of the chemical shifts of the indole nitrogens suggests that all eight of them are hydrogen bonded. Using difference spectroscopy, we isolate several changes in these hydrogen bonds in the early and late M states. As found earlier for the peptide backbone, some perturbations found in the early M state relax in the transition to the late M state while new perturbations arise. Interestingly, Rotational Echo DOuble Resonance (REDOR) difference spectroscopy of [20-13C]retinal,[indole-15N]Trp-bR shows that indole of Trp182 is not involved in the significant hydrogen bond perturbations. We also use REDOR to measure dipolar interactions in [20-13C]retinal,[indole-15N]Trp-bR, and thereby determine the distance between the C20 of retinal and the indole nitrogen of Trp182. The internuclear distance changes only slightly from the light-adapted state (3.36 +/- 0.2 A) to the early M state (3.16 +/- 0.4 A).
Petkova
Aneta T
AT
Department of Chemistry and Keck Institute for Cellular Visualization, MS #015, Brandeis University, Waltham, Massachusetts 02454-9110, USA.
Hatanaka
Minoru
M
Jaroniec
Christopher P
CP
Hu
Jingui G
JG
Belenky
Marina
M
Verhoeven
Michiel
M
Lugtenburg
Johan
J
Griffin
Robert G
RG
Herzfeld
Judith
J
D001436:Bacteriorhodopsins / Q000737:chemistry*; D002247:Carbon Isotopes; D007211:Indoles; D008027:Light; D008956:Models, Chemical; D009587:Nitrogen Isotopes; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods; D017494:Proton Pumps / Q000737:chemistry; D018717:Purple Membrane / Q000737:chemistry; D012172:Retinaldehyde / Q000737:chemistry; D014364:Tryptophan / Q000737:chemistry*
D002247:Carbon Isotopes; D007211:Indoles; D009587:Nitrogen Isotopes; D017494:Proton Pumps; D001436:Bacteriorhodopsins; D014364:Tryptophan; D012172:Retinaldehyde