Detection of fast light-activated H+ release and M intermediate formation from proteorhodopsin.
BACKGROUND
Proteorhodopsin (pR) is a light-activated proton pump homologous to bacteriorhodopsin and recently discovered in oceanic gamma-proteobacteria. One perplexing difference between these two proteins is the absence in pR of homologues of bR residues Glu-194 and Glu-204. These two residues, along with Arg-82, have been implicated in light-activated fast H+ release to the extracellular medium in bR. It is therefore uncertain that pR carries out its physiological activity using a mechanism that is completely homologous to that of bR.


RESULTS
A pR purification procedure is described that utilizes Phenylsepharose and hydroxylapatite columns and yields 85% (w/w) purity. Through SDS-PAGE of the pure protein, the molecular weight of E.-coli-produced pR was determined to be 36,000, approximately 9,000 more than the 27,000 predicted by the DNA sequence. Post-translational modification of one or more of the cysteine residues accounts for 5 kDa of the weight difference as measured on a cys-less pR mutant. At pH 9.5 and in the presence of octylglucoside and diheptanoylphosphotidylcholine, flash photolysis results in fast H+ release and a 400-nm absorbing (M-like) photoproduct. Both of these occur with a similar rise time (4-10 micros) as reported for monomeric bR in detergent.


CONCLUSIONS
The presence of fast H+ release in pR indicates that either different groups are responsible for fast H+ release in pR and bR (i.e. that the H+ release group is not highly conserved); or, that the H+ release group is conserved and is therefore likely Arg-94 itself in pR (and Arg-82 in bR, correspondingly).
Krebs
Richard A
RA
Chemistry Department, Syracuse University, Syracuse, NY 13244-4100, USA. rakrebs@syr.edu
Alexiev
Ulrike
U
Partha
Ranga
R
DeVita
Anne Marie
AM
Braiman
Mark S
MS
D003545:Cysteine / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D006863:Hydrogen-Ion Concentration; D017136:Ion Transport; D007700:Kinetics; D008027:Light; D008970:Molecular Weight; D009154:Mutation; D017494:Proton Pumps / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D011522:Protons; D012243:Rhodopsin / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D025604:Rhodopsins, Microbial; D013057:Spectrum Analysis
D017494:Proton Pumps; D011522:Protons; D025604:Rhodopsins, Microbial; C413548:proteorhodopsin; D012243:Rhodopsin; D003545:Cysteine