PMC:103662 / 4479-12401 JSONTXT

{"target":"https://pubannotation.org/docs/sourcedb/PMC/sourceid/103662","sourcedb":"PMC","sourceid":"103662","source_url":"https://www.ncbi.nlm.nih.gov/pmc/103662","text":"Results\n\nPurification\nPR was obtained in 85% purity, assuming that values of ε280 and ε546 for pR are the same as for bR solubilized in DMPC/cholate/SDS mixtures at pH 8 (ε280= 7.85 × 104 cm-1 M-1 and ε551 = 4.8 × 104 cm-1 M-1) [5]. This assumption is actually expected to underestimate the purity of pR produced, by up to ~20%, since the pR we expressed has 10 tryptophan and 14 tyrosine residues, as compared to 8 tryptophans and 11 tyrosines in bR from H. salinarum. The absorbance of contaminant proteins was assumed to be 1.1 for a 1 mg/mL solution. By using these assumptions, the relative concentrations of pR and other proteins can be determined from the absorbance spectra of the various fractions (fig. 1). The resulting purity values correlate well with those Coomasie-stained SDS-PAGE gels (see below). The OG extract of cholate-washed membrane pellets starts out at a pR content of 7% total protein (w/w). The Phenylsepharose column increases the purity level to 24%, with approximately 5% loss. The final purification step by hydroxylapatite column chromatography produces pR with ~85% purity and a further loss of ~60%, i.e. the overall yield of the two column procedure was ~30%.\nFigure 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.\n\nPolyacrylamide gel electrophoresis\nRelative to protein standards, the apparent molecular weight of bR is 25,000 while the apparent molecular weights of pR-wt and pR-TCM are 36,000 and 31,000, respectively (fig. 2, lanes E and C, respectively). SDS-PAGE (fig. 2) also confirms the estimates of purity level based on the assumed ε280/ε546 ratio identical with that of detergent solubilized bR. Interestingly, the pR appears to be a doublet band whose relative concentrations remain almost unchanged during purification. This doublet is also present in the less-purified sample of pR-TCM, with both bands shifted down by approximately the same amount (fig. 2, lane C).\nFigure 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. Subsequent SDS-PAGE analysis of pR samples that had been stored for periods of time up to several months indicate that after sitting for several weeks in octylglucoside solution at 4°C, the largest post-translational modification on wild-type pR is eliminated – presumably hydrolysed off of the cysteine(s) – leaving only a 31,000-MW band indistinguishable from that seen for pR-TCM (data not shown). Furthermore, after boiling for several min in gel loading solution, this cleaved wild-type protein, as well as the TCM, both give an extra artifactual band near 36,000 dalton. The latter band, a singlet, is coincidentally at almost the same apparent MW as the doublet from the uncleaved post-translationally-modified wild type pR (fig. 2, lane E). These potential artifacts should be taken into consideration in any attempt to reproduce the results in Fig. 2.\n\nPhotocycle kinetics and flash-induced proton concentration changes\nPhotocycle kinetics were measured at 400, 500. and 580 nm in the presence of the short-chain lipid DHPC. This lipid does not support the formation of closed bilayer vesicles, but rather forms micelles like a detergent. The time-resolved measurements showed no positive 400-nm absorbance signals at pH 8.0 or lower (Fig. 3). This is somewhat in disagreement with Béjà et al [1], who detected small 400-nm transient absorbance increases upon photolysis at pH 8.0. However, we observed a transient 400-nm absorbance increase at an elevated pH of 9.5 (fig. 3).\nFigure 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. At pH 9.5 in the presence of DHPC, and observing transient changes at 500 nm (fig. 4), pR undergoes a 2-phase decay after the initial unresolved absorbance decrease. Multiexponential fits show that the first decay phase has a time constant of 4 μs, in good agreement with the 4-μs rise time of the 400 nm signal (Fig. 4). The amplitude of this decay represents about 80% of the initial absorbance depletion. The second phase of the 500 nm absorbance decay occurs with a substantially slower time constant of 0.5 s, returning the remaining 20% of initial absorbance change. The slowest decay components of the positive 400-nm signal and the negative 500-nm signal follow similar kinetics, although the amplitudes of these components differ by a factor of 3. At pH 9.5, the 580 nm trace has no significant positive values indicative of an O-like intermediate, although, in agreement with earlier measurements [1], at lower pH values a red-shifted transient is the predominant positive absorbance signal (data not shown).\nFigure 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 shows a different type of time-resolved measurement, probing not the pR chromophore, but rather pH changes in the protein environment. Proton concentration changes in the aqueous bulk phase were measured with the pH sensitive dye cresol red, which has a pKa of 8.2–8.5. The bottom trace in Figure 5 shows the absorbance change of the indicator during the pR photocycle. The negative signal is indicative of a pH decrease, corresponding to transient H+ release from the protein into the solution. The best-fit time constant for the release phase is 6 μs. The positive 400 nm trace in fig. 5 (reproduced from fig. 3) shows that the proton release and uptake follow kinetics very similar to the apparent formation and decay of M, as is typically seen in bR near neutral pH [6,7,21]. However, no proton release signal could be observed for pR at pH 6 or 8 (data not shown).\nFigure 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.","divisions":[{"label":"title","span":{"begin":0,"end":7}},{"label":"sec","span":{"begin":9,"end":1625}},{"label":"title","span":{"begin":9,"end":21}},{"label":"p","span":{"begin":22,"end":1195}},{"label":"figure","span":{"begin":1196,"end":1625}},{"label":"label","span":{"begin":1196,"end":1204}},{"label":"caption","span":{"begin":1206,"end":1625}},{"label":"p","span":{"begin":1206,"end":1625}},{"label":"sec","span":{"begin":1627,"end":3695}},{"label":"title","span":{"begin":1627,"end":1661}},{"label":"p","span":{"begin":1662,"end":2292}},{"label":"figure","span":{"begin":2293,"end":2834}},{"label":"label","span":{"begin":2293,"end":2301}},{"label":"caption","span":{"begin":2303,"end":2834}},{"label":"p","span":{"begin":2303,"end":2834}},{"label":"p","span":{"begin":2835,"end":3695}},{"label":"title","span":{"begin":3697,"end":3763}},{"label":"p","span":{"begin":3764,"end":4320}},{"label":"figure","span":{"begin":4321,"end":4746}},{"label":"label","span":{"begin":4321,"end":4329}},{"label":"caption","span":{"begin":4331,"end":4746}},{"label":"p","span":{"begin":4331,"end":4746}},{"label":"p","span":{"begin":4747,"end":5765}},{"label":"figure","span":{"begin":5766,"end":6310}},{"label":"label","span":{"begin":5766,"end":5774}},{"label":"caption","span":{"begin":5776,"end":6310}},{"label":"p","span":{"begin":5776,"end":6310}},{"label":"p","span":{"begin":6311,"end":7189}},{"label":"label","span":{"begin":7190,"end":7198}}],"tracks":[]}