High resolution 13C-solid state NMR of bacteriorhodopsin: assignment of specific aspartic acids and structural implications of single site mutations

Abstract

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.

Asp85 is the only internal aspartic acid that gets protonated in the M intermediate and the purple‐to‐blue transition of bacteriorhodopsin A solid‐state13C CP‐MAS NMR investigation

“…In addition, tbJ¢ mutant, D96N, has the identical chromophore as ~iild.type BR both in the ground state and M [26]. In 0~ur earlier NMR studies [21,22] the spectra of the wildt~pe and the mutant, DgsN, did not differ as regards the it~,Jternal Asp or the Trp resonances. Therefore it is rea-,~ol~able to assume that the conclusions drawn for the ~ssidues Asp ss and Asp :b' also hold true for wild-type l}I~R.…”

“…Three pronoun~d siti~nals are found in the ground state. They have forl~¢rly been assigned to the deprotonated Asp ~-~ (176.3 R)~m) and Asp ~s (173.7 ppm) as well as to the protonated i~sp ~ (170.3 ppm) [20][21][22]. These assignments made ~ls~ of FTIR studios [5] which showed that Asp b ~s is the ~ccnd internal protonated asparti¢ acid in the BR ~r~and state.…”

“…Two protonateat (Asp ~, As# ts) and two deprotonatcd (Asp sS, Asp ~2) aspartic acids wore observed in the BR groundstat• [20][21][22] …”

The reaction cycle of bacteriorhodopsin: an analysis using visible absorption, photocurrent and infrared techniques

“…% shows positive bands at 1765 cm-1 and 1755 cm-a which are characteristic for the deprotonation of Asp residues. Since independent evidence suggests that Asp212 and Asp85 are deprotonated in the ground state this rate must be correlated to the reformation of bR (Gerwert et al 1989;Braiman et al 1988;Engelhard et al 1989Engelhard et al , 1990Siebert et al in preparation): In earlier work using time resolved IR techniques it has been shown that the absorption changes at 1765 cm 1 and 1755 cm -~ are kinetically different and it was argued that these signals are due to two Asp residues which are deprotonated in the ground state Engelhard et al 1985). Since the sign of the amplitudes between 1770 cm-~ and 1745 cm-1 is positive this band is due to a deprotonation and this must necessarily succeed signals which can be explained by a protonation, such as z 2 and z 4.…”

The early development and application of FTIR difference spectroscopy to membrane proteins: A personal perspective

“…The results from the research described above on BR as well as a variety of other spectroscopic studies [7,10,39,63,82,108,139,161,162,225]) led by 1990 to the "standard" model of the BR proton pump mechanism" shown in Fig. 13.…”