Magic angle sample spinning carbon-13 nuclear magnetic resonance of isotopically labeled bacteriorhodopsin

Abstract

Bacteriorhodopsin (bR), the light-driven proton pump protein from Halobacterium halobium, was biosynthetically labeled with [4-13C]Asp. The incorporation yield was 48%. The magic angle sample spinning (MASS) 13C nuclear magnetic resonance (NMR) spectrum of this sample revealed six different peaks superimposed on a broad band of naturally abundant peptide-bond 13C. Two of the six carbonyl signals can be attributed to internal-protonated Asp carboxyl groups, one of which might be Asp115. An additional resonance at 110 ppm can be associated with the C-11 carbon of Trp, indicating an unusual biosynthetic pathway of this amino acid in Halobacterium halobium. Similar measurements performed on papain-treated purple membrane which lacks the C-terminal tail display two new intense signals at 178 and 178.9 ppm. If the same spectrum is taken without cross-polarization, these signals do not decrease or disappear. On the basis of their intensities and their chemical shifts, one can assign in addition to the C-terminal Asp four Asp residues facing the cytoplasmic phase. In native bR, at least two of these form a salt-bridge-like bond which also might include the C-terminal tail. These experiments not only provide data about the chemical environment of the Asp residues within the hydrophobic core of bacteriorhodopsin but also yield information about the interactions between surface components.

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

“…Again this can be explained by a protonation of Asp ss resulting in a new band at 169.5 ppm with an almost identical o~o as that found in the M intermediate. The signals of Asp ~ and Asp bzs now merge into one broad band due to the shift of the resonance of Asp ~s from 170.3 to 171 ppm, as previously shown [20]. Both residues are still protonated.…”

“…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.…”

“…1 shows difference spectra of[4-~3C]Asp-labelled~aiaas native PM obtained from the light-adapted ~0~nd state and the M intermediate. The cross polarization technique favours the internal groups which are i~ a rather rigid environment [20]. Three pronoun~d siti~nals are found in the ground state.…”

“…*;C]Asp was incorporated into bacteriorhodopsin by growing the bacteria in a synthetic medium [10,20] and purple membrane was isolated following the standard proc~ures [24]. Wild-tyl~ bacteriorhodopsin was deionized by a cation-exchange reagent leading to the so-called blue BR which had an absorption maximum of 602 nm.…”

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