5. Results and Discussion
5.1.1. Dynamics for Bacteriorhodopsin in native purple membranes
The native environment of Bacteriorhodopsin is the purple membrane. In this environment the protein has been extensively studied. The proton transfer dynamics are well-known and step-scan FTIR spectroscopy contributed strongly in deciphering the individual proton transfer steps and revealing the molecular details of the proton transfer of Bacteriorhodopsin [9, 90–96].
In this project, the purified Bacteriorhodopsin in purple membrane served as reference sample to establish the step-scan setup and optimize the measurement parameters. The measured spectra were used for comparison with published data found in the literature.
Figure 5.1 shows a 3D plot of measured step-scan FTIR difference spectra of purified BR in purple membrane. The measurements were performed with a time-resolution of 10µs and an acquisition range from 1000 cm-1 to 3900 cm-1 in a time range from 10 µs to 19 ms. Vibrations of the protein are located between 1000 cm-1 and 1900 cm-1. The expansion of the acquisition range to 3900 cm-1 had the intention to be able to monitor also the region between 2800 cm-1 and 3000 cm-1 in which the vibrations of the lipid alkyl chains can be monitored. Unfortunately, the strong water absorption between 3000 cm-1 and 3500 cm-1 interferes with the lipid alkyl chain region. Attempts to overcome this problem using D2O as a solvent or deuterated lipid chains are discussed in section 5.1.6.
The 3D plot shows for every time slice the corresponding difference absorption spectrum and gives therefore an overview about the infrared spectral changes taking place during the photocycle of Bacteriorhodopsin. The recorded spectra coincide well with published literature data [87].
5. Results and Discussion
Figure 5.1.: Time-resolved absorbance changes for BR in purple membrane recorded with an acquisition range from 1000 cm-1to 3500 cm-1.
Protonation dynamics of acidic amino acids are probed in the carboxylic region between 1700 cm-1 and 1800 cm-1 where the carboxyl side chain is well isolated from other vibrations of the protein [19]. Kinetics of the protonation of the primary proton acceptor Asp85 can be followed at 1760 cm-1 as visualized in a contour plot in Figure 5.2.
Figure 5.2.: Contour-plot of the carboxylic region. Protonation changes of the primary proton acceptor Asp85 are observed at 1760 cm-1. Positive absorbance changes are color-coded in red and negative absorbance changes in blue.
5.1. Bacteriorhodopsin
Figure 5.3 shows the 10 µs and 500 µs difference spectrum between 1000 cm-1 and 3500 cm-1 and an amplification between 1000 cm-1 and 1900 cm-1 which is the protein region of interest. The absorbance changes of the C-C retinal stretching vibration at 1188 cm-1 in the fingerprint region can be regarded as an indicator of the photocycle dynamics [92]. A positive band describes the retinal in 13-cis isomerization together with a protonated Schiff base [82]. The decay and rise of this band over time shows the deprotonation and subsequent reprotonation of the Schiff base. In the 10µs difference spectrum this band is clearly visible, whereas in the 500µs difference spectrum this band decays, indicating the deprotonation of the Schiff base. The corresponding time trace of this band is shown in Figure 5.4. The minimum of this time trace is at ~500µs, when the Schiff base is deprotonated during the photocycle. The proton which is released by the Schiff base protonates the primary proton acceptor Asp85. The band at 1760 cm-1 is characteristic for the protonation dynamics of Asp85 [97]. For the 10 µs difference spectrum in Figure 5.3 no positive band is observed at 1760 cm-1, while for the 500 µs difference spectrum a positive band rises. The intensity change can be monitored revealing the protonation of Asp85. For the later intermediates N and O, the C=O stretching vibration of Asp85 shifts by approximately 5 cm-1 down to 1755 cm-1. The band at 1760 cm-1is therefore equivalent to measuring the kinetics of the M intermediate [98]. The time trace of this band is depicted in Figure 5.4. The rise of the band is characteristic for the rise of the M intermediate which is equivalent to the protonation of Asp85 and the following decay corresponds to the decay of the M intermediate. The maximum of the time trace at 1760 cm-1 correlates with the minimum of the time trace at 1188 cm-1 at ~500µs, revealing the same time course for the first proton transfer step and reflecting the direct proton transfer from the Schiff base to Asp85 [99]. The time traces coincide well with previously published time traces measured with time-resolved FTIR spectroscopy [87]. A biexponential fit was used to fit the dynamics of the time traces, withτ1 representing the dynamics of the primary proton transfer step and τ2 the slower dynamics of the M intermediate decay. A time constant ofτ1 ∼86 µs was derived for the protonation of Asp85 for the fit of the time trace at 1760 cm-1, coinciding with a time constant derived for the deprotonation of the Schiff base (τ1 ∼85 µs) and fit of the time trace at 1188 cm-1. In the literature, the rise of the kinetics at 1760 cm-1 is assigned with a half-life of∼ 60 µs [87] which corresponds to a time constant of ∼ 87 µs, matching with our obtained results.
5. Results and Discussion
1760
1760
Figure 5.3.: Time-resolved difference spectra recorded at 10µs (blue) and 500µs (black) after light excitation for BR in native purple membrane measured at room temperature.
Upper panel: acquisition range from 1000 cm-1 to 3500 cm-1. Lower panel:
magnification of the protein region of interest from 1000 cm-1 to 1900 cm-1.
5.1. Bacteriorhodopsin
Figure 5.4.: Time-traces revealing the protonation dynamics of Asp85 monitored at 1760 cm-1 (left) and the Schiff base monitored at 1188 cm-1 (right) for BR in purple membrane. Rise of the curve at 1760 cm-1reports on the protonation of Asp85 (rise of the M intermediate), the following decay on the decay of the M intermediate.
Decay of the curve at 1188 cm-1 reports on the deprotonation of the Schiff base and following rise on its reprotonation. Both time-traces are fitted with a biexponential function (red). The maximum of the fit at 1760 cm-1 occurs
~500 µs, likewise the minimum of the fit at 1188 cm-1 which implies that the Schiff base is fully deprotonated when Asp85 is protonated. This shows the direct proton transfer from the Schiff base to Asp85. Deprotonation of the Schiff base and protonation of Asp85 occur with the same time constants.