High resolution 13C-solid state NMR of bacteriorhodopsin: assignment of specific aspartic acids and structural implications of single site mutations.
Three mutant strains of Halobacterium sp. GRB with the site of mutation in the bacterioopsin gene (PM 326: Asp96----Asn; PM 374: Asp96----Gly; PM 384: Asp85----Glu) were grown in a synthetic medium containing (4-13C)-Asp. The mutant bacteriorhodopsins labeled with (4-13C)-Asp (37%-45%), and owing to the metabolism of Halobacteria also with (11-13C)-Trp (50%-100%), were isolated as purple membranes and 13C Solid State Magic Angle Sample Spinning (MASS) Nuclear Magnetic Resonance (NMR) spectra of the samples were taken. The Asp96 mutants lacked the signal at 171.3 ppm which was previously assigned to a protonated internal Asp (Engelhard et al. 1989a). This observation supports the conclusion that Asp96 is protonated in the ground state. PM 384 (Asp85----Glu) has an absorption maximum at 610 nm. It can be converted into a purple form (lambda max = 540 nm) by treatment with a detergent (CHAPSO). The NMR-spectra of these two species differ from each other and from the wild type. The intensity of the resonance at 173 ppm in the wild type spectrum is reduced in both forms of the mutant protein. It is probable that this signal is caused by Asp85. The amino acid changes result not only in a perturbation of their direct environment but also effects on Trp residues and the chromophore protein interaction can be observed.
Engelhard
M
M
Max-Planck-Institut für Ernährungsphysiologie, Rheinlanddamm, Dortmund, Federal Republic of Germany.
Hess
B
B
Metz
G
G
Kreutz
W
W
Siebert
F
F
Soppa
J
J
Oesterhelt
D
D
D001224:Aspartic Acid; D001436:Bacteriorhodopsins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D002247:Carbon Isotopes; D005971:Glutamates; D018698:Glutamic Acid; D005998:Glycine; D006217:Halobacterium / Q000378:metabolism; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D009154:Mutation; D014364:Tryptophan
D002247:Carbon Isotopes; D005971:Glutamates; D001224:Aspartic Acid; D018698:Glutamic Acid; D001436:Bacteriorhodopsins; D014364:Tryptophan; D005998:Glycine