PMC:103662 / 1714-4477
Annnotations
{"target":"https://pubannotation.org/docs/sourcedb/PMC/sourceid/103662","sourcedb":"PMC","sourceid":"103662","source_url":"https://www.ncbi.nlm.nih.gov/pmc/103662","text":"Background\nProteorhodopsin is a 249-amino acid membrane protein native to several uncultured species of γ-proteobacteria, which are a component of marine plankton [1]. Addition of retinal to E. coli expressing pR was shown to cause a reddish coloration of the bacteria with an absorption maximum near 520 nm. The pR contained in the bacterial membranes was shown to act as a light-activated proton pump, but only when retinal is present. Time-resolved UV/vis studies at pH 8 also revealed that the protein undergoes a photocycle, similar to that of wild type bacteriorhodopsin, but with a predominance of the O intermediate instead of M.\nThe bR photocycle has been characterized by spectroscopic methods as having six principal photointermediates: bR, K, L, M, N and O. Each intermediate has a distinct absorbance maximum; the most studied are bR (570 nm), M (412 nm), and O (640 nm) since these are the ones that can be produced in the highest concentration at physiological pH values. Monitoring of the absorbance at individual wavelengths after photoexcitation is used to determine the relative concentrations and decay times of each of these photointermediates. The L → M transition in bR is characterized by the deprotonation of the Schiff base to Asp-85, producing the distinctive 412 nm absorbance maximum of M, and by so-called fast proton release, the ejection of a proton from a different (unknown) residue into the external medium on the ~10–100 μs time scale, depending on pH. Reprotonation of the Schiff base from Asp-96 occurs during the M → N transition with an absorbance maximum of 560 nm [2]. The N → O transition involves the reprotonation of the Asp-96 from the cytoplasmic space.\nLike bR, pR consists of seven transmembrane α-helices that include in the membrane interior all of the residues conserved among archaeal rhodopsin proton pumps. In particular, analogues of Asp-85, Asp96, Arg-82, and Lys-216 of bR are present in pR. Conspicuously absent are analogues for Glu-194 and Glu-204 of bR. The latter, as well as Arg-82, have been implicated in fast proton release. In particular, mutagenesis of Glu-194 or Glu-204 in bR results in loss of fast proton release [3,4]. The absence of homologs for these residues in pR leaves open the question of whether it carries out fast H+ release.\nExperiments described here demonstrate that pR does indeed undergo fast H+ release, at least under elevated pH conditions that resemble somewhat those of the γ-proteobacteria's native open ocean environment. We also demonstrate that there is a post-translational modification of at least one of the three native cysteines when pR is expressed in E. coli. Both of these discoveries were made possible through purification methods for pR described herein.","divisions":[{"label":"title","span":{"begin":0,"end":10}},{"label":"p","span":{"begin":11,"end":637}},{"label":"p","span":{"begin":638,"end":1700}},{"label":"p","span":{"begin":1701,"end":2309}}],"tracks":[]}