5. Results and Discussion
5.1.6. Simultaneous probing of lipid and protein dynamics
Apart from investigating the influence of the physical properties of the lipid environment on the function of the protein, another goal of this project was to explore simultaneously the dynamics of the surrounding lipids. Assignments for the vibrations of phospholipids are listed in section 4.5. Vibrations of the phospholipid headgroups (1050-1250 cm-1) and the ester vibration (~1732 cm-1) at the interface between headgroup and lipid chain interfere with vibrations of the protein (1000-1800 cm-1), whereas lipid chain vibrations do not interfere with vibrations of the protein. Therefore, the focus was set on identifying dynamics of the lipid chain vibrations. However, the broad absorption of water between 3000 cm-1 and 3500 cm-1 interferes with the weak lipid chain vibrations between 2800 cm-1 and 3000 cm-1 hindering a reliable analysis. The artifact of the broad water absorption arises from a laser-induced transient temperature rise [88].
Hydrogen-Deuterium Exchange
In a first attempt, a hydrogen-deuterium exchange (H-D exchange) was performed for BR in purple membrane by centrifuging the sample and resuspending it several times in D2O and heating up the sample between the centrifugation steps to 65 ◦C. The purpose was to shift the water absorption out of the frequency region of the lipid vibrations.
The dried sample on the calcium fluoride window was then resuspended with D2O accordingly. Figure 5.20 shows the absorption spectra of the BR in D2O sample as well as the respective time-resolved difference spectra. The frequency-shifted D2O absorption band is observed between 2250 cm-1 and 2750 cm-1, but residual H2O still remains and is observable by the absorption band between 3000 cm-1 and 3500 cm-1. The monitored decrease of absorption of the amide II band at 1547 cm-1 and increase of absorption at 1461 cm-1 is characteristic for H-D exchange due to NH to ND exchange of peptide backbone groups [103]. The absorption of Asp85 carboxylic side chain vibration shifts also from 1760 cm-1 for COOH to 1751 cm-1 for COOD [103]. Kinetics of the primary proton transfer step displayed in Figure 5.21 reveal a significant slowdown of the primary proton transfer step. The minimum at 1188 cm-1, maximum at 1751 cm-1 respectively occurs delayed compared to BR in H2O at ~2 ms (compare with kinetics of BR in H2O in subsection 5.1.1). These kinetic results are in good agreement with kinetic data for the rise and decay of the M intermediate measured with transient visible spectroscopy at 412 nm found in the literature for BR in D2O [104, 105]. However, the main purpose of H-D
5.1. Bacteriorhodopsin
exchange was to identify dynamics of the lipid chain vibrations. In the time-resolved difference spectra (5.20) the very strong absorption of D2O and residual absorption of H2O which interfere on both sides of the region of the lipid chain vibrations, does not allow conclusions on possible small difference bands of lipid chain dynamics.
3500 3000 2500 2000 1500 1000
0.0 0.5 1.0 1.5 2.0
A/OD
wavenumber / cm -1
2959 2926
2870
1657 1547
1461
Figure 5.20.: Upper panel: Absorption spectrum of BR in D2O. A strong absorption band of D2O is observed between 2250 cm-1 and 2750 cm-1. Residual H2O is also observable between 3000 cm-1and 3500 cm-1. Lower panel: Step-scan FTIR difference spectra of BR in D2O.
5. Results and Discussion
Figure 5.21.: BR in D2O time traces at 1751 cm-1(left) and 1188 cm-1(right). The maximum at 1751 cm-1, minimum at 1188 cm-1, respectively occurs delayed at ~2 ms as compared to BR in H2O.
Dynamics of lipids and protein reconstituted with deuterated lipids
In order to overcome the difficulties of strong H2O or D2O absorption interfering in the region of lipid chain vibrations, lipids with deuterated chains were selected which shift the lipid chain vibrations in the region of 2000 cm-1 to 2200 cm-1 which is free from the absorption of any other groups. Possible small difference bands in this region can then unequivocally be assigned to lipid chain dynamics. BR was therefore reconstituted into 18:0 PC-d70 liposomes (section 4.2). 18:0 PC-d70 lipids are structurally equivalent to DSPC, with the only difference that all the carbons of the lipid chains are deuterated.
Figure 5.22 demonstrates the absorption spectrum of BR in 18:0 PC-d70 liposomes and respective time-resolved step-scan difference spectra. Deuterated lipid chain bands are visible in the absorption spectrum at 2196 cm-1, 2154 cm-1 and 2087 cm-1, but also residual hydrated lipid chain bands still occur at 2961 cm-1, 2924 cm-1 and 2854 cm-1. In the time-resolved difference spectra, small difference bands clearly show up in the deuterated lipid chain region at 2195 cm-1, 2155 cm-1 and 2091 cm-1.
5.1. Bacteriorhodopsin
2195 2155
2091
1763
1187 2961
2924 2854
2196 2087
2154
1656
1547 1735
2091 2155 2195
Figure 5.22.: Upper panel: Absorption spectrum of BR reconstituted with deuterated lipids.
Lower panel: Step-scan FTIR difference spectra of BR reconstituted with deuterated lipids. The inset shows a magnification for the deuterated lipid chain spectral region between 2000 cm-1 and 2250 cm-1.
The coresponding time traces of these deuterated lipid chain difference bands are displayed in Figure 5.23. All three difference bands show a similar dynamical behaviour:
a strong absorption change at the beginning of the measurement at 10µs which decays
5. Results and Discussion
over time. However, between roughly 400µs and 800µs, the absorbance change first rises again before it finally decays back to zero. Interpretation of this dynamical behaviour is on a speculative level. The huge absorbance change at the beginning of the measurement and following decay might likely be a heat artifact due to the laser excitation pulse heating up the sample [88]. The rise of the absorbance change between 400 µs and 800 µs could be interpreted as a dynamic response of the lipid chains due to conformational changes of the protein during the M intermediate. The oscillations observable in the signal due to fluctuations of the mirror position are mainly present in the millisecond time-scale [88], but it cannot be excluded that the rise of the absorbance change between 400 µs and 800 µs is also part of these oscillations and not driven by protein-lipid interactions.
The protonation dynamics of the primary proton transfer step for BR in the deuterated lipid chain environment depicted in Figure 5.24 are similar and comparable to the BR in DSPC protonation dynamics with hydrated lipid chains. For Schiff base deprotonation, a time constant of 75 µs is obtained and for Asp85 protonation a time constant of 82µs.
Figure 5.23.: Lipid chain dynamics for BR reconstituted with deuterated lipids at 2195 cm-1 (left), 2155 cm-1 (middle) and 2091 cm-1(right).
Figure 5.24.: Protonation dynamics for BR reconstituted with deuterated lipids at 1760 cm-1 (left) and 1188 cm-1 (right).
5.1. Bacteriorhodopsin
5.1.7. Steady-state measurements
In order to further explore deuterated lipid chain difference bands, steady-state measure-ments were performed as introduced in section 4.8. For BR, a mixture of the M and N intermediate can be monitored by cooling the sample to -5◦C and continuous light >
495 nm has to be applied. The M/N-BR difference spectrum can be calculated, when measuring an absorption spectrum in the dark (BR) and an absorption spectrum after 5 min of continuous exposure to light > 495 nm (M/N). Figure 5.25 displays the M/N-BR difference spectrum of BR in purple membrane (red spectrum).
3500 3000 2500 2000 1500 1000
-5
Figure 5.25.: Steady-state measurements of BR in purple membrane (-5 ◦C, > 495 nm continuous light illumination). The red spectrum shows the M/N-BR (light minus dark) difference spectrum and the black spectrum shows the BR-BR (dark minus dark) difference spectrum used as control.
The positive band at 1760 cm-1 clearly indicates that a significant amount of protein is
5. Results and Discussion
observed in the M intermediate. The noise level is visualized by subtracting an absorption spectrum measured in the dark by an absorption spectrum measured also in the dark (black spectrum). Without light excitation, no protein difference bands are observed.
An increased noise level is observed in the spectral region of broad water absorption (3000 cm-1-3500 cm-1) as well as atmospheric CO2 (2300 cm-1-2400 cm-1).
As a next step, steady state measurements were performed on BR reconstituted into 18:0 PC-d70 liposomes. The M/N difference spectrum (red spectrum) in Figure 5.26 displays the same protein difference bands characteristic for a significant amount of BR in the M intermediate as shown above for BR in purple membrane.
2191 2090
Figure 5.26.: Steady-state measurements of BR reconstituted into 18:0 PC-d70 liposomes (-5 ◦C, > 495 nm continuous light illumination). The red spectrum shows the M/N-BR (light minus dark) difference spectrum and the black spectrum shows the BR-BR (dark minus dark) difference spectrum. Difference bands of the deuterated lipid chains occur at 2191 cm-1and 2090 cm-1. The inset shows a magnification for the spectral region between 2000 cm-1 and 2250 cm-1.
Moreover, small difference bands at 2191 cm-1 and 2090 cm-1 can be monitored which
5.1. Bacteriorhodopsin
can be clearly assigned to the deuterated lipid chain environment. The dark minus dark spectrum (black spectrum) reveals neither protein nor deuterated lipid difference bands.
The deuterated lipid chain difference bands seem to correlate with the rise of the M/N intermediate. However, in part of the measurements, difference bands in the deuterated lipid chain region occured also in the dark minus dark control spectra. Sample equilibrium and temperature stability are relevant factors which have to be controlled precisely to ensure that the deuterated lipid chain difference bands arise due to protein excitation.
Control spectra give the opportunity to determine the temperature and sample stability of the measurement which is a prerequisite to evaluate the reliability of the identified weak lipid difference bands.