3 Time-resolved spectroscopy of protein function

Transcription

1 3 Time-resolved spectroscopy of protein function Milian Wolff and David Ehrenberg Freie Universität Berlin (Supervised by Bernd Schultz and Victor Lorenz Fonfria) (Dated: June 25, 213) In this experiment the kinetics of the photocycle of Bacteriorhodopsin (br) is investigated with two techniques, UV/Vis flash photolysis and step-scan FT-IR difference spectroscopy. The goal is to get an insight into the proton transport functionality of br in purple membrane (PM), by classifying the different photointermediates and and measuring the proton uptake from and release to the bulk solution. Flash photolysis provided five photointermediates, which were distinguishable, while the data obtained via Fourier transform infrared spectroscopy (FTIR) yielded three spectrally different intermediates. Furthermore rate constants were obtained by fitting the kinetics at certain wavenumber with exponential decay functions. The results are in agreement with those reported in literature ([1], [2]). By adding pyranin to the sample, which serves as a ph indicator, it allowed a qualitative analysis of the number of protons involved in the photocycle. FIG. 1. Photocycle of br (taken from Wilmouth et al. [3]). FIG. 2. The proton transfer during the photocycle of br (taken from Neutze et al. [5]). INTRODUCTION The protein br acts as a proton pump, i.e. it enables protons to move from the intercelluar to the intracelluar area of a cell. The resulting proton gradients is used for ATP synthase. For this process the br absorbes green light (5 to 65 nm) with a maximum at 568 nm. Thus it converts the energy coming from the light to chemical energy. During this process the br undergoes several intermediate states, which is schematically shown in fig. 1. Each state has its characteristic structual changes and maximum absorption wavelength. After absorbing light, the retinal of the br photoisomerizes from all-trans to 13-cis. This results in the intermediate J. The critical steps in the photocycle are the deand reprotonation of the Schiff base (see fig. 2).During the transition from K to L the hydrogen bonding between the Schiff base and the Asp-85 on the intercelluar side. A proton of the Schiff base gets released during the transition from L to M. The Schiff base it now deprotonated. In order to get reprotonated it gets a proton from Asp-96 during the transition to N. Now the retinal goes back to all-trans (N to O) and finally the deprotonation of the Aps-85 finishes the photocycle [4]. The focus of this experiment is a deeper understanding of the photocycle of glsbr. For this purpose step scan FTIR and flash photolysis (FP) will be used. Both techniques deliver time resolved spectra which is required in order to follow the photocycle. In addition these techniques are complementary. While the side chains of br absorb in the infrared and undergo vibrational transitions, the retinal is absorbing in the range of visible light where electron transitions take place. Hence, combining these it is possible to follow roughly the position of the proton during its pumping.

2 2 METHODS & MATERIALS Flash Photolysis Flash Photolysis (FP) is a pump probe technique. In this experiment a ND:YAG laser was used for pumping with a second harmonic generator in order to get a wavelength of 532 nm. Since the br has its absorption maximum at 568 nm the laser should excite a good amount of photocycles. For probing a 1 W halogen lamp with a monochromator was used to get asborption spectra for different wavelengths. Since the kinetics will be plotted on a logarithmic scale it is appropiate to record many datapoints in first decades and just a few in the last decades. For this purpose two oscilloscopes were used, which recorded in 4 ns and 124 ns intervals, respectively. The two resulting curves were then merged with a given program. In order to increase the signal-to-noise ratio several repitions were recorded and then averaged. Time Resolved FTIR There exist two techniques for getting a time resolved spectra with FTIR. Rapid scan, which is simply moving the mirror rapidly back and forth. Thus the resolution is determined by the speed of the moving mirror. The other technique, which is used in this experiment, is step scan. For each position of the mirror a full photocycle is recorded, i.e. for a specific duration. The spacing between the sampling positions determines the resolution of the bandwidth while the time resolution is limited by the rise time of the IR detector or, more practical, by the digitalization speed of the converter. For pumping the second harmonic of the ND:YAG laser was used. The data were postprocessed with the method of single value decomposition (SVD) as described in Lorenz-Fonfria and Kandori [1]. This was done with a program written in MatLab. Sample Preparation Kinetic Measurements via Flash Photolysis (FP): For optical measurements it is beneficial to have a sample with an Optical density (OD)(57) of around.7. There, the absorption still gives a good signal and is still in the linear region where Lambert-Beer s law holds [6]. A concentration of.3 mg /ml of br corresponds to a OD(57) of ca..7. To obtain 1 ml of such a solution, 5 µl of br (c = 6 mg /ml) was diluted with 1 µl of buffer (KH 2 PO 4 /K 2 HPO 4, c = 5 mm) and 5 µl of salt (KCl, c = 15 mm) in 8 µl of Milli-Q water. Thus the sample should have an OD(57) of about.7, with 5 mm bufferand 15 mm salt concentration. Proton Uptake From Bulk Solution: In order to get information about the proton concentration pyranin served as a ph indicator. Analogous to above a 2 ml br sample with a theoretical OD(57) of.7 was prepared from 269 µl stock solution of br with OD(57) of 5.2, 15 mm KCl (1 µl, 5 mm) and filled up with Milli- Q. It was directly observable that the sample had a too high OD due to a false valuation of the stock solution. Thus the sample solution was diluted to 4 ml by adding 5 µl of KCl and 195 µl water. The ph was adjusted using a solution of 3 mm KOH to a value of about 7.3. The sample solution was then divided into two cuvettes of 1 ml and in one 3.5 µl of 14 mm pyranin was added. The other used samples used in this experiment were prepared beforehand. RESULTS UV/Vis Flash Photolysis Sample Verification To measure the OD an initial steady-state UV/Vis spectrum was taken and is shown in 3. The leftmost feature stems from aromatic amides in the protein backbone. The small peak in the middle arises from free retinal in the solution. The rightmost feature finally is due to retinal bound in br. The peak maximum shows that the desired OD of about.7 was not reached, but rather a less opaque solution with an OD of ca..5 was prepared. This is still in line with the expectations. From Lambert-Beer s law A = ɛ c d, (1) with A being the absorbance, ɛ the extinction coefficient, c the concentration and d the path length, one can now deduce the br concentration of the sample. At λ = 53 nm the extinction coefficient is known to be ɛ = 63 mol 1 cm [Lab Course Script]. The path length is 1 cm, i.e. the thickness of the cuvette. Thus with A 53 nm.4 this yields a concentration of c 6.4 µmol. TODO: mol is keine konzentration! Kinetics Subsequently the kinetics of br between 4 nm to 63 nm were measured in 1 nm increments via flash photolysis. The combined results are shown in fig. 4. Note how the range between 5 nm to 6 nm gives an overview over the whole photocycle: The difference absorption drops considerably, due to the conformational changes away from the initial dark-adapted br state with its characteristic peak at around 57 nm (cf. fig. 1).

3 optical density OD [-] wavelength λ [nm] FIG. 3. UV/Vis spectrum of br in buffer solution; peaks from left to right: aromatic amides in the protein backbone (28 nm), free retinal (4 nm), bound retinal (57 nm) FIG. 4. kinetics of br between 4 nm to 63 nm in 1 nm increments Eventually after a total time span in the order of about 5 ms all br molecules in the sample have completed the photocycle and returned into the dark-adapted br state. As one can see, it is very hard to classify the K, L and N photointermediates. Their characteristic wavelengths of maximum absorption, i.e. 59, 55 nm and 56 nm respectively, lie relatively close together and are furthermore superimposed with changes due to dark-adapted br, which decays initially and builds up with the finish of the photocycle. Thus a rough interpretation only gives insight into the creation of the M intermediate, which is clearly separated from the other photointermediates. The evaluation of the kinetics there, as shown in fig. 5c, yields a time constant of about 65 µs for the creation and 4.3 ms for the decay of the M intermediate. This is in conformance with the values of 35 µs to 56 µs for the transition of L M 1 and 5. ms to 7. ms for M 2 N reported by Lorenz-Fonfria and Kandori [1]. A more in-depth peak analysis of the kinetics at λ = 59 nm (cf. fig. 5a), i.e. where one expects to see the K intermediate, yields comparable data. The dominant decay in the kinetic curve relates to the creation of the L M 1 state, this time with a time constant of about 73 µs. The shoulder arises from the K L transition, with a time constant of ca. 1 µs which is comparable to the value of 6.3 µs to 9.8 µs reported by Lorenz-Fonfria and Kandori [1]. The interpretation of the final ascent is ambiguous though. Its time constant of 5.5 ms can be related to either the M 2 N transition, or arises from the end of the photocycle, where br transitions back into the dark adapted state. Lorenz-Fonfria and Kandori [1] list values of 5. ms to 7. ms and 6.9 ms to 8.8 ms for these two transitions. Finally it is possible to observe the O intermediate, which is also relatively isolated (cf fig. 1, 5e). A fit of the final feature, i.e. starting at a time of about 3 µs, yields a time constant of about 3.3 ms for the ascent and 5. ms for the decay. This is compatible with 2. ms to 3. ms for N O and 6.9 ms to 8.8 ms for O BR [1]. An in-depth analysis of the kinetics for L (fig. 5b) and N (fig. 5d) are omitted. The fact that they initially start at a negative difference absorption hints at the fast nature of the initial transition away from the br darkadapted state to J and K. Otherwise, one again only sees the transition to M and then to either N or BR. The transitions to L or N are not clearly observable. Proton Transport Finally one can investigate the proton transport functionality of br in PM with two new samples. Both have a ph of about 7.3 initially and do not contain a buffer. One sample furthermore contains pyranine that acts as a ph indicator. This then makes the proton transport visible, as the PM patches swim freely in the solution and no transport between clearly separated areas takes place. Rather protons are temporarily bound to br during the photocycle and eventually released once it has finished. A steady state UV/Vis spectrum was recorded for both samples and is shown in fig. 6. The pyranine-free solution shows the same characteristics as described above in sec.. The addition of pyranine gives rise to its two characteristic peaks in the visible spectrum at 44 nm and 454 nm. The peak at 37 can be ignored. Comparison to the pyranine-free sample also shows that the pyranine affects the other peaks by reducing their OD. This is taken into account in the following by scaling the results of the pyranine sample by a factor of 1.15, which is obtained

4 e 91e 81e 71e 61e e 91e 81e 71e 61e e 91e 81e 71e 61e (a) K 59, λ = 59 nm (b) L 55, λ = 55 nm (c) M 412, λ = 41 nm e 91e 81e 71e 61e e 91e 81e 71e 61e (d) N 56, λ = 56 nm (e) O 64, λ = 64 nm FIG. 5. kinetics of br for characteristic wavelengths of photointermediates optical density OD [-] wavelength λ [nm] without pyranine with pyranine FIG. 6. UV/Vis spectrum of br without buffer solution for sample with and without pyranine from the ratio of the peak OD related to the retinal in br at about 57 nm. Comparison of the OD of the characteristic pyranine peaks allows the deduction of the ph value of the sample by comparison to literature values [Lab Course Script]. This then confirms the ph value of about 7.3 for a ratio of about 1.4 between the peak OD. Now the kinetics of the two samples were measured via flash photolysis at the two characteristic pyranine wavelengths of 44 and 454 nm. Subtraction of the curve of the pyranine-free sample allows to isolate the pyranine kinetics which are shown in fig. 7. Note that the peaks do not align on the time axis, which is unexpected. One assumes that the incline of the 44 nm peak is due to an increase of the OH concentration in the pyranine solution, while the decline of 454 nm is attributed to a decrease of the H 3 O + concentration. Thus these peaks should change synchronously. It is unclear where this artifact comes from. Nonetheless one can see that the OD 44 increases maximally by ca..8 whereas the OD 454 decreases by ca..4. Taking the absolute data of fig. 6 into account, this can be related to a ph change to roughly 7.1 for an OD ratio of about 1.6. Be aware though that the ph value could only be read of rather coarsely from the source. Still, this should allow for a qualitative analysis of the amount of protons that take part in the photocycle via N p ( ) mol l 1 1 ml = 29.3 pmol. (2) Compared to the br concentration of ca µmol (cf. eq. (1), with OD 53.83) this is only a small fraction in the order of 2.2 ppm. Finally one can investigate the time constants of the kinetics, which are roughly 13 µs and 6 ms. The former is quite large when compared with the value given by Lorenz-Fonfria and Kandori [1] for the L M 1 transition of 35 ms to 56 ms. The latter though is in good agreement with the 5. ms to 7. ms given for the M 2 N transition. These transitions are those where the conformational changes lead to a deprotonation/protonation of the Schiff Base.

5 nm 454 nm (a) raw data.2.4 1e 9 1e 8 1e 7 1e 6 1e FIG. 7. kinetics of pyranine at characteristic wavelengths of 44 nm and 454 nm Step-scan FT-IR difference spectroscopy (b) data SVD Data Processing In order to reduce the noise, a Single value decomposition (SVD) was performed. Hereby a data matrix D(nν nt ) is factorized into D = U S VT. (3) U(nν nt ) contains the orthonormal basis spectra, S(nτ nt ) is a diagonal matrix consisting of the singular values and V(nt nt ) is the abstract time-trace matrix, where nν is the number of wavelengths/frequency points and nt is the number of time points. Since the photocycle of br consists of a finite number of spectrally distinct intermediates, it is possible with the help of this technique not only to reduce the noise but also to identify how many intermediates are clearly distinguishable [7]. This was realized by a program written in Matlab by the tutor. This program also adds some modifications which are explained in [1]. For the decomposition a number of four photointermediates were chosen, since it was not expected to see the K intermediate because of its short conformation time ( ps) [8]. The results are presented in fig. 8 and show nicely the noise reduction. Difference Spectra of the Intermediates In comparison to the work of [9] the intermediates of the photocycle were extracted out of the time-resolved data shown in fig. 8. The difference absorption spectra, in respect to dark-adapted br, are shown in fig. 9. The maximum concentration of the L intermediate was found at 6.25 µs. As an indicator served the peak at 1189 cm 1, (c) filtered residuals (d) weighted filtered residuals FIG. 8. kinetics of br in step-scan FTIR, logscale time axis in µs which corresponds to C C stretch of the retinal [1]. This peak is characteristic for the L intermediate and since it decays completely in the M intermediate, it is a good indicator. The M intermediate, which maximum concentration was found after µs, was identified by the peak at 1762 cm 1. This peak is also present in the N intermediate, but shifted to smaller wavenumbers. In addition the peak at 1185 cm 1 served as an indicator for

6 FIG. 1. Expansion of the side chain region of the difference spectra presented in fig. 9. The lines indicate specific side groups FIG. 9. Difference spectra for the L (red, 6.24 µs), M (blue, µs) and N (green, 4737 µs) intermediate. The indicated peaks were chosen to identify the different intermediates. Negative peaks correspond to ground state br, while positive peaks indicates vibrational modes of the photointermediates. this state. Because of the kinetic degeneracy of the reset function, the O intermediate is not clearly distinguishable of the long-living intermediate M and N [9]. The photocycle was finished after.1 s and the br returned into its ground state. Despite of the difficulty to identify the maximum concentration of one intermediate, the estimated times are in agreement with results presented in works done by Riesle et al. [1] and Radu et al. [11]. The bands between 1211 cm 1 and 1164 cm 1 correspond to the chromophore, between 1581 cm 1 and 1499 cm 1 to its backbone, between 1671 cm 1 and 165 cm 1 to the backbone of amide-i [12]. Because the focus of this work is on the proton transfer via the side chains, the region between 18 cm 1 and 17 cm 1 is of particular interest [11] and is presented in fig. 1. To examine if a side group is protonated or not, its essential to note that the deprotonated from of an amino acid (see fig. 11) is not IR-active in this range in contrast to the C C stretch vibration. The band at 1762 cm 1 has been assigned to Asp-85, the primary acceptor of the proton FIG. 11. An amino acid in its (1) protonated and (2) deprotonated forms coming from the Shiff base ([9], [11]). Thus it can be concluded that Asp-85 is protonated in the M intermediate. This peak shifts to 1754 cm 1 in the N intermediate, due to a different environment, indicating that Asp-85 is still protonated. The proton donor for the Shiff base Aps- 96 is deprotonated in N-intermediate, indicated by the negative band at 1741 cm 1. Determination of Rate Constants In order to determine the rate constants of the photocycle the kinetics at wavenumber 1186 cm 1 were chosen and is shown in fig. 12. This decision was made because it is expected to see the decay of all the intermediates at this wavenumber. Since only three intermediates were distinguishable in this experiment, a fit with three exponential decay function was performed in Origin. The results are listed in tab. I. The obtained values correspond to the results reported by Souvignier and Gerwert [2]. This underlines the assumption that only the L, M and N intermediates are spectrally present in the time resolved FTIR spectrum.

7 7.8 CONCLUSION.6 TODO.4 absorption A [-] e 6 1e FIG. 12. Kinetics of 1186 cm 1. TABLE I. Fit results of a three exponential decay function of the kinetics at 1186 cm 1. Transition Rate constant L M 99 ± 3 µs M N 2.2 ±.1 ms N br 31 ± 4 ms mail@milianw.de david.ehrenberg@fu-berlin.de [1] V. A. Lorenz-Fonfria and H. Kandori, Journal of the American Chemical Society 131, 5891 (29). [2] G. Souvignier and K. Gerwert, Biophysical journal 63, 1393 (1992). [3] R. C. Wilmouth, I. J. Clifton, and R. Neutze, Natural product reports 17, 527 (2). [4] J. K. Lanyi, Biochimica et Biophysica Acta (BBA)- Bioenergetics 1757, 112 (26). [5] R. Neutze, E. Pebay-Peyroula, K. Edman, A. Royant, J. Navarro, and E. M. Landau, Biochimica et Biophysica Acta (BBA) - Biomembranes 1565, 144 (22), membrane Protein Structure. [6] Freie Universität Berlin, 3 Vibrational Spectroscopy Applied to Biomolecules, (213). [7] C. Gergely, L. Zimanyi, and G. Váró, The Journal of Physical Chemistry B 11, 939 (1997). [8] J. Heberle, Biochimica et Biophysica Acta (BBA)- Bioenergetics 1458, 135 (2). [9] C. Zscherp and J. Heberle, The Journal of Physical Chemistry B 11, 1542 (1997). [1] J. Riesle, D. Oesterhelt, N. A. Dencher, and J. Heberle, Biochemistry 35, 6635 (1996). [11] I. Radu, M. Schleeger, C. Bolwien, and J. Heberle, Photochemical & Photobiological Sciences 8, 1517 (29). [12] K. Gerwert, G. Souvignier, and B. Hess, Proceedings of the National Academy of Sciences 87, 9774 (199).