Detection of fast light-activated H+ release and M intermediate formation from proteorhodopsin

Abstract

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).

Figure 1

UV/visible absorption spectra of pR in octylglucoside solution (1–3%) at three stages of purification. All three spectra were measured in the presence of octylglucoside at pH 8, and are normalized to the 280-nm protein peak. Spectrum A, the OG extract of cholate-washed E. coli membranes; spectrum B, pooled 546-nm absorbing fractions from Phenylsepharose column; spectrum C, same material after hydroxylapatite column.

Figure 2

SDS-PAGE of pR (wild type and pR-triple cysteine mutant). Lane A contains bacteriorhodopsin (bR). Lanes B and F contain BioRad protein molecular weight markers including labeled bands at 21.5 (trypsin inhibitor), 31 (bovine carbonic anhydrase), and 45 (ovalbumin) kDa. Lane C is of the pR triple cysteine mutant (TCM). Lane D contains the Phenylsepharose™-purified pR wild type protein, corresponding to spectrum B of fig. 1. Lane E contains the hydroxylapatite-purified pR wild type protein, corresponding to spectrum C in fig. 1.

Figure 3

Dependence on pH of the M-like intermediate of pR. Time courses of flash-induced absorbance changes measured at 400 nm and 22°C for pR in 1% DHPC/100 mM NaCl solution at pH 6.5, 8.0 and 9.5. A positive differential absorbance at 400 nm is indicative of the presence of the M intermediate. The logarithmic time scale ranges from 10⁰–10⁷ μs after photolysis by a 10-ns laser pulse at 500 nm, with an energy of 3–6 mJ.

Figure 4

Photocycle kinetics of pR at selected wavelengths at pH 9.5. Time traces were measured at 400, 500, and 580 nm. The 400-nm trace shows the kinetics of the M intermediate, i.e. the deprotonated Schiff base, as in Fig. 3. The 500-nm trace shows the depletion signal of pR at the earliest times, and then the time course of the N intermediate as well as return of the pR resting state. The 580-nm trace is indicative of an O-like intermediate. The conditions are 1% DHPC, 100 mM NaCl, pH9.5 at 22°C. The laser excitation is as in fig. 3.

Figure 5

Comparison of the kinetics of M formation and decay with kinetics of ET release and uptake. The time trace of the M-like intermediate was measured at 400 nm (upper panel). Time-resolved H⁺ concentration changes (lower panel) were measured with the pH indicator dye Cresol Red. A negative Cresol Red absorbance change at 580 nm is indicative of a transient decrease in the pH of the solution, i.e. of H⁺ release by pR. Solid lines represent multiexponential fits, with the main rise and decay times indicated for the M intermediate. The H⁺ release and uptake time constants obtained from the fit are marked with arrows pointing down for release and pointing up for uptake. Sample and excitation conditions are as in fig. 4.