Rot mutiert zu Rosa: Während bisher angenommen wurde, dass nur die Aminosäuren nahe des Retinalchromophors maßgeblich für die Farbabstufung durch Rhodopsine sind, wurde nun gefunden, dass beim Proteorhodopsin, einem Archaea‐Rhodopsin aus Meeresbakterien, eine Farbänderung aus der Mutation von Ala178 zu Arg in der E‐F‐Schleife resultiert. Die Rotverschiebung beträgt im neutralen pH‐Bereich ungefähr 20 nm (siehe Bild).

Bacteriorhodopsin (BR), a membrane protein found in Halobacterium salinarum, functions as a light-driven proton pump. The Schiff base region has a quadrupolar structure with positive charges located at the protonated Schiff base and Arg82, and the counterbalancing negative charges located at Asp85 and Asp212. The quadropole inside the protein is stabilized by three water molecules, forming a roughly planar pentagonal cluster composed of these waters and two oxygens of Asp85 and Asp212 (one from each carboxylate side chain). It is known that BR lacks proton-pumping activity if Asp85 or Asp212 is neutralized by mutation, but binding of Cl- has different functional effects in mutants at these positions. Binding of Cl- to D85T converts into a chloride ion pump (Sasaki, J., Brown, L. S., Chon, Y.-S., Kandori, H., Maeda, A., Needleman, R., and Lanyi, J. K. (1995) Science 269, 73-75). On the other hand, photovoltage measurements suggested that binding of Cl- to D212N restores the proton-pumping activity at low pH (Moltke, S., Krebs, M. P., Mollaaghababa, R., Khorana, H. G., and Heyn, M. P. (1995) Biophys. J. 69, 2074-2083). In this paper, we studied halide-bound D212N mutant BR in detail. Light-induced pH changes in a suspension of proteoliposomes containing D212N(Cl-) at pH 5 clearly showed that Cl- restores the proton-pumping activity. Spectral blue-shift induced by halide binding to D212N indicates that halides affect the counterion of the protonated Schiff base, whereas much smaller halide dependence of the lambdamax than in D85T suggests that the binding site is distant from the chromophore. In fact, the K minus BR difference Fourier-transform infrared (FTIR) spectra of D212N at 77 K exhibit little halide dependence for vibrational bands of retinal and protein. The only halide-dependent bands were the C=N stretch of Arg82 and some water O-D stretches, suggesting that these groups constitute a halide-binding pocket. A strongly hydrogen-bonded water molecule is observed for halide-bound D212N, but not for halide-free D212N, which is consistent with our hypothesis that such a water molecule is a prerequisite for proton-pumping activity of rhodopsins. We concluded that halide binding near Arg82 in D212N restores the water-containing hydrogen-bonding network in the Schiff base region. In particular, the ion pair formed by the Schiff base and Asp85 through a strongly hydrogen-bonded water is essential for the proton-pumping activity of this mutant and may be controlled by the halide binding to the distant site.

It is usually assumed that only amino acids located near the retinal chromophore are responsible for color tuning of rhodopsins. However, we recently found that replacement of Ala178 with Arg in the E-F loop of proteorhodopsin (PR), an archaeal-type rhodopsin in marine bacteria, shifts the lambda(max) from 525 to 545 nm at neutral pH [Yoshitsugu, M., Shibata, M., Ikeda, D., Furutani, Y., and Kandori, H. (2008) Angew. Chem., Int. Ed. 47, 3923-3926]. Since the location of Ala178 is distant from the retinal chromophore (approximately 25 A), the molecular mechanism of the unusual mutation effect on color tuning is intriguing. Here we studied this mechanism by using additional mutations and some analytical methods. Introduction of Arg into the corresponding amino acid in bacteriorhodopsin (BR, M163R mutant) does not change the absorption spectra, indicating that the effect is specific to PR. Introduction of Arg into the A-B or C-D loop yields little (3 nm) or no color change, respectively. T177R and P180R mutants exhibited absorption spectra identical to that of the wild type, while N176R and S179R mutants exhibit lambda(max) values of 528 and 535 nm, respectively. Therefore, the observed color change is position-specific, being fully effective at position 178 and half-effective at position 179. Salt affects the absorption spectra of wild-type and A178R PR similarly. FTIR spectroscopy at 77 K indicated similar chromophore structures for wild-type and A178R PR, and A178R PR pumps protons normally. We infer that the E-F loop has a unique structure in PR and the mutation of Ala178 disrupts the structure that includes the transmembrane region, leading to the observed changes in color and pK(a).

Switching from red to pink: It has been believed that only the amino acids near the retinal chromophore are responsible for the color tuning of rhodopsins. Herein is reported a color change of proteorhodopsin, an archaeal‐type rhodopsin in marine bacteria, by mutation of Ala178 to Arg at the E–F loop. The red shift at a neutral pH value amounts to 20 nm (see graph).

No abstract

No abstract

No abstract

No abstract