5. Results and Discussion
5.2.1. Dynamics for Proteorhodopsin reconstituted into liposomes
The obtained findings of measuring time-resolved FTIR data on BR-membrane interaction were used and adapted in a next step to investigate Proteorhodopsin, whose function and mechanism is far less understood than for Bacteriorhodopsin. Proteorhodopsin can change proton pumping vectoriality depending on pH, introduced in subsection 2.3.3.
Proteorhodopsin in DDM detergent was gratefully received from the collaborating group of Clemens Glaubitz, Goethe University Frankfurt. All measurements were performed at pH 7.0 except for the measurements in subsection 5.2.5.
The obtained sample was reconstituted as a first step into DOPC liposomes. Figure 5.29 displays the time-resolved absorbance changes of the photocycle measured from 1000 cm-1 to 1900 cm-1 in a time range from 20 µs to 37960 µs.
Figure 5.29.: Time-resolved absorbance changes for PR in DOPC liposomes recorded with an acquisition range from 1000 cm-1 to 1900 cm-1in a time range from 20 µs to 37960µs.
A lower time-resolution of 20 µs was selected due to the overall slower dynamics
5. Results and Discussion
for Proteorhodopsin compared to Bacteriorhodopsin. Maximal absorbance changes (C=C retinal stretching vibration at 1522 cm-1) were recorded on the order of 1 to 2 mOD. The quantum yield of Proteorhodopsin (0.5) is estimated to be lower than for Bacteriorhodopsin (0.7) [106] which might explain the reduced recorded signal intensity.
The recorded light-induced absorbance changes for PR in DOPC are in good agreement with the only two previously published time-resolved step-scan spectra of Proteorhodopsin in a mixture of lipids (65% DOPC, 20% DOPE, 10% DOPS and 5% cholesterin or polar lipids of the purple membrane)[36] or of PR in DMPC [107]. The maximal absorbance changes of these studies were also on the order of around 1 mOD or less. Figure 5.30 shows selected time-resolved difference spectra at 20µs and 2000 µs.
1900 1800 1700 1600 1500 1400 1300 1200 1100 1000 -2.5
Figure 5.30.: Time-resolved absorption difference spectra recorded at 20µs (blue) and 2000µs (black) after light excitation for PR in DOPC measured at room temperature,
pH 7. Assignments are based on [36, 107] and discussed in the text.
Bands were assigned according to the above cited publications of Friedrich et al. [36]
and Xiao et al. [107] and are discussed in the following. The negative C-C bands of the retinal stretching vibrations occur at 1161 cm-1, 1200 cm-1, 1233 cm-1 and 1253 cm-1. Except for the band at 1233 cm-1, these bands are all also found for Bacteriorhodopsin.
The latter band is assumed to be coupled to the N-H in-plane bending mode of the retinal Schiff base. The positive band at 1188 cm-1 is indicative of the C14-C15 stretching
5.2. Proteorhodopsin
vibration of the retinal in the K intermediate. The C=C stretching vibration of the ground state appears at 1522 cm-1. A strong positive band of the C=C stretch at 1508 cm-1 is observable in the 20µs difference spectrum which is indicative of the K intermediate.
This positive band is for Bacteriorhodopsin no longer present when measuring with microsecond time-resolution and becomes only visible when measuring with nanosecond time-resolution (compare subsection 5.1.8). The decay of the K-like intermediate is slower for Proteorhodopsin and can be monitored with microsecond time-resolution. At later times, the C=C stretch of the photoproduct shifts to higher wavenumbers to 1547 cm-1. This C=C retinal vibration at 1547 cm-1 overlaps with amide II conformational changes of the protein which report on major global structural changes of the protein. The positive band rising up in the 2000 µs difference spectrum at 1755 cm-1 is indicative of the protonation of the primary proton acceptor Asp97. Assignment of this band has been confirmed previously with mutation studies of Asp97 [108]. The contour plot in Figure 5.31 visualizes the measured absorbance changes in the carboxylic region. A rise and decay of the band at 1755 cm-1 is monitored similar to the 1760 cm-1 band of Asp85 for Bacteriorhodopsin, but with altered protonation dynamics. Contrary to Bacteriorhodopsin, the band does not shift at later intermediates. The small negative band at 1722 cm-1 is assigned to the C=C stretch of the primary proton donor Glu108, but dynamics of this band cannot easily be resolved due to low signal intensities.
Figure 5.31.: Contour-plot of the carboxylic region. Protonation changes of the primary proton acceptor Asp97 are observed at 1755 cm-1. Positive absorbance changes are color-coded in red and negative absorbance changes in blue.
5. Results and Discussion
The time-resolved dynamics of selected bands are displayed in Figure 5.32. The rise and decay of the band at 1755 cm-1 shows the protonation and subsequent deprotonation dynamics of Asp97. With the maximum taking place around 5 ms, protonation dynamics of Asp97 are significantly slowed down as compared to protonation dynamics of Asp85 of BR in purple membrane (maximum at ~500µs). Due to the slower dynamics, analysis of the time traces was performed by evaluating the maxima of triexponential fits in this case.
The band at 1188 cm-1of the C14-C15retinal stretch decays in two phases. The first decay until approximately 3 ms is probably associated with the decay of the K intermediate and Schiff base deprotonation, but in contrast to the corresponding band for BR, no further rise of this band is observed which could be associated with reprotonation of the Schiff base. Instead, in form of a second decay, the band relaxes back to the ground state. The band at 1188 cm-1 for BR is assigned to be positive, when the Schiff base is protonated and simultaneously the retinal is in 13-cis conformation [82]. A possible explanation for the second decay at 1188 cm-1 for PR might be, that at these later times the retinal already starts to relax back to all-trans conformation and therefore the reprotonation of the Schiff base can no longer be tracked at this wavenumber. These changes in the retinal conformation and the ground state recovery kinetics can be monitored at 1527 cm-1 [98].
The kinetics of the retinal start to relax back slowly at approximately 1 ms, but at 10 ms there is still a significant amount of the retinal in 13-cis conformation. Therefore, the hypothesis that the reprotonation of the Schiff base can not be tracked due to an already relaxed all-trans retinal might not hold true and further investigations are needed how to interprete the dynamics of the 1188 cm-1 band for PR. Due to the slower photocycle dynamics of PR, the decay of the K intermediate can be followed with microsecond time-resolution at 1508 cm-1. The large positive band at the beginning of the acquisition time of 20 µs decays over time and disappears at approximately 1 ms. The main global changes of the protein can be tracked by the changes of the amide II band of the protein monitored at 1547 cm-1 and will be discussed in more detail in subsection 5.2.4.
5.2. Proteorhodopsin
1547 cm-1 1755 cm-1
1527 cm-1 1508 cm-1
1188 cm-1
Figure 5.32.: Selected time traces of PR in DOPC at 1755 cm-1 (protonation dynamics of Asp97), 1188 cm-1(interpretation of this band not yet clear and further discussed in main text), 1527 cm-1 (C=C retinal of the unphotolyzed state, ground state recovery kinetics), 1508 cm-1 (decay of the K intermediate) and 1547 cm-1 (amide II conformational changes and C=C retinal in N intermediate).
5.2.2. Variation of the lipid properties
Similar to subsection 5.1.3 for BR, the same lipids (except for DMoPC) were used for reconstitution of PR into proteoliposomes to investigate the influence of the lipid physical properties on the protonation dynamics of PR. In contrast to BR, the native lipid environment of PR is not accurately known, since PR has been originally discovered by genomic screening analysis of sea water [10, 68]. However, theE.coli membrane has a
5. Results and Discussion
similar lipid composition as the marine bacteria, in which PR was first identified [68]. The E.coli membrane consists of only three different phospholipids, phosphatidylethanolamine (80%), negatively charged phosphatidylglycerol (15%) and cardiolipin (5%), with an average lipid chain length of more than 16 carbon atoms and about half of the lipids being monounsaturated [109]. A variation of these lipid physical properties could therefore be performed with the lipids used in this study with proximity and applicability to the native membrane environment of PR. Corresponding time-resolved difference spectra can be found in section C in the appendix.
Lipid chain length
PR was reconstituted into DMPC (14:0) and into DSPC (18:0) liposomes to investigate the influence of the lipid chain length on the protonation dynamics. Figure 5.33 displays the protonation dynamics of Asp97 monitored at 1755 cm-1 for the two different lipid environments.
100 1000 10000
0.0 0.2 0.4 0.6 0.8 1.0 1.2
PR in DSPC
PR in DMPC
A(normalized)/a.u.
time / µs
Figure 5.33.: Protonation dynamics of Asp97 for PR in DMPC (14:0) and PR in DSPC (18:0).
Apart from small differences during the protonation of Asp97, the recorded time traces showed similar dynamical behaviour. For PR in DSPC, the maximum of the time trace was evaluated to occur at 8676µs, for PR in DMPC at 11740µs , respectively. Due to the
5.2. Proteorhodopsin
low signal-to-noise ratio obtained for the PR measurements, the accuracy of these results should be handled with care. Reassuring kinetics from the Schiff base deprotonation correlating with protonation of Asp97 cannot be provided, since interpretation of the band at 1188 cm-1 is still questionable (compare subsection 5.2.1). Nevertheless, the results suggest that a variation of the lipid chain length for 14 and 18 carbon chain length does not highly affect and alter the protonation dynamics of the primary proton transfer step of PR.
Degree of saturation
In order to investigate the effect of degree of saturation on the protonation dynamics, PR was reconstituted into DSPC (18:0) and into DOPC (18:1) liposomes, respectively (Figure 5.34).
100 1000 10000
0.0 0.2 0.4 0.6 0.8 1.0 1.2
PR in DOPC
PR in DSPC
A(normalized)/a.u.
time / µs
Figure 5.34.: Protonation dynamics of Asp97 for PR in DSPC (18:0)and PR in DOPC (18:1).
For PR in DOPC, the maximum was determined to take place at 4152 µs, whereas for PR in DSPC the maximum was obtained at 8676µs. The faster dynamics recorded for the DOPC lipid environment indicate that the higher disorder of the liquid phase of the DOPC lipids facilitates and enhances the protonation dynamics of the primary proton transfer step of PR. The observed faster kinetics for the protonation of Asp97 would
5. Results and Discussion
coincide with the monitored faster protonation of Asp85 for BR in DOPC (compare 5.1.3).
However, no longer-lived M intermediate is observable for PR in DOPC as compared to BR in DOPC. Instead, a faster deprotonation of Asp97 for PR in DOPC is observed compared to PR in DSPC.
Photocycle kinetics for PR in nanodiscs are reported to be accelerated for longer, unsaturated alkyl chains of POPC as compared to DMPC [69]. These findings fit well to the observations for BR in nanodiscs, showing faster photocycle kinetics for DOPC as compared to DMPC [67]. The exclusive variation of the degree of saturation in the here presented results shows that the unsaturated alkyl chains are the important factor for the observed altered kinetics. The lipid phase is therefore for BR and PR influencing the photocycle kinetics.
Charge of lipid headgroups
Figure 5.35 shows the protonation dynamics of Asp97 for PR reconstituted into DMPG liposomes having negatively charged lipid headgroups and PR reconstituted into DMPC / DMTAP 1:1 liposomes having positively charged lipid headroups due to the DMTAP
lipids.
Figure 5.35.: Protonation dynamics of Asp97 for PR in DMPG (14:0,-)and PR in DMPC (14:0) / DMTAP (14:0,+) 1:1.
5.2. Proteorhodopsin
Table 5.2.: Time of maximum of primary proton transfer step dynamics for Proteorhodopsin reconstituted into the different lipid environments.
Sample Maximum in µs at 1755 cm-1
PR in DSPC 8676 µs
PR in DMPC 11740 µs
PR in DOPC 4152 µs
PR in DMPG 9160 µs
PR in DMPC/DMTAP 1:1 14142 µs
The time traces indicate a low influence of the different lipid charges on the protonation dynamics of Asp97. PR has in contrast to BR an asymmetric charge density (Figure C.10 in Appendix), with the C-terminal having an overall positive charge and the N terminal containing negatively charged residues [110]. Due to the poor signal-to-noise ratio it might be likely that possible effects of charged headgroups on the protonation dynamics could not be resolved.