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FTIR difference spectroscopy of bacteriorhodopsin: Toward a molecular model (1992)

Rothschild, Kenneth J.

Springer

Journal of bioenergetics and biomembranes 24 (1992), S. 147-167

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ISSN: 1573-6881

Keywords: Proton transport ; biomembranes ; infrared ; membrane protein ; energy transduction

Source: Springer Online Journal Archives 1860-2000

Topics: Biology , Chemistry and Pharmacology , Physics

Notes: Abstract Bacteriorhodopsin (bR) is a light-driven proton pump whose function includes two key membrane-based processes, active transport and energy transduction. Despite extensive research on bR and other membrane proteins, these processes are not fully understood on the molecular level. In the past ten years, the introduction of Fourier transform infrared (FTIR) difference spectroscopy along with related techniques including time-resolved FTIR difference spectroscopy, polarized FTIR, and attenuated total reflection FTIR has provided a new approach for studying these processes. A key step has been the utilization of site-directed mutagenesis to assign bands in the FTIR difference spectrum to the vibrations of individual amino acid residues. On this basis, detailed information has been obtained about structural changes involving the retinylidene chromophore and protein during the bR photocycle. This includes a determination of the protonation state of the four membrane-embedded Asp residues, identification of specific structurally active amino acid residues, and the detection of protein secondary structural changes. This information is being used to develop an increasingly detailed picture of the bR proton pump mechanism.

Type of Medium: Electronic Resource

URL: http://dx.doi.org/10.1007/BF00762674

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Vibrational spectroscopy of bacteriorhodopsin mutants: I. Tyrosine-185 protonates and deprotonantes during the photocycle (1988)

Braiman, Mark S. ; Mogi, Tatsushi ; Stern, Lawrence J. ; [et al.]

New York, NY : Wiley-Blackwell

Proteins: Structure, Function, and Genetics 3 (1988), S. 219-229

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ISSN: 0887-3585

Keywords: proton transport ; energy transduction ; purple membrane ; proton wire ; Schiff base counter-ion ; Chemistry ; Biochemistry and Biotechnology

Source: Wiley InterScience Backfile Collection 1832-2000

Topics: Medicine

Notes: The techniques of FTIR difference spectroscopy and site-directed mutagenesis have been combined to investigate the role of individual tyrosine side chains in the proton-pumping mechanism of bacteriorhodopsin (bR). For each of the 11 possible bR mutants containing a single Tyr→Phe substitution, difference spectra have been obtained for the bR→K and bR→M photoreactions. Only the Tyr-185→Phe mutation results in the disappearance of a set of bands that were previously shown to be due to protonation of a tryosinate during the br→K photoreaction [Rothschild et al.: Proceedings of the National Academy of Sciences of the United states of America 83:347, (1986)]. The Tyr-185→Phe mutation also eliminates a set of bands in the bR→M difference spectrum associated with deprotonation of a Tyr; most of these bands (e.g., positive 1272-cm-1 peak) are completely unaffected by the other ten Tyr→Phe mutations. Thus, tyrosinate-185 gains a proton during the bR→K reaction and loses it again when M is formed. Our FTIR spectra also provide evidence that Tyr-185 interacts with the protonated Schiff base linkage of the retinal chromophore, since the negative C=NH+ stretch band shifts from 1640 cm-1 in the wild type to 1636 cm-1 in the Tyr-185→Phe mutant. A model that is consistent with these results is that Tyr-185 is normally ionized and serves as a counter-ion to the protonated Schiff base. The primary photoisomerization of the chromophore translocates the Schiff base away from Tyr-185, which raises the pKa of the latter group and results in its protonation.

Additional Material: 4 Ill.