Single-shot submicrosecond mid-infrared spectroscopy on protein reactions with quantum cascade laser frequency combs

Transcription

1 Supporting Information for Single-shot submicrosecond mid-infrared spectroscopy on protein reactions with quantum cascade laser frequency combs Jessica L. Klocke, Markus Mangold,*, Pitt Allmendinger, Andreas Hugi, Markus Geiser, Pierre Jouy, Jerome Faist, and Tilman Kottke Physical and Biophysical Chemistry, Department of Chemistry, Bielefeld University, Universitätsstr. 25, Bielefeld, Germany. IRsweep AG, Laubisruetistrasse 44, 8712 Staefa, Switzerland. Institute for Quantum Electronics, ETH Zurich, 8093 Zurich, Switzerland. Table of Contents: Figure S1: Global fit of results from dual-comb spectroscopy at ph 7.2 Figure S2: Global fit of results from step-scan FTIR spectroscopy Figure S3: Representiveness of a single shot Figure S4: Influence of the probe light exposure on the sample Figure S5: Infrared absorption spectra of bacteriorhodopsin samples with different path lengths Figure S6: Comparison of dual-comb experiments on samples with different path lengths Figure S7: Global fit of results from dual-comb spectroscopy at different ph values Figure S8: Global fit of a single shot from dual-comb spectroscopy and standard deviations Supporting references S1

2 Figure S1: Global fit of results from dual-comb spectroscopy at ph 7.2. (A) The concentration profiles of the intermediates were obtained by applying a kinetic model of sequential first-order reactions with four intermediates. The fitted profiles (straight lines) show some statistical deviations from the experiment (points), in particular for the two early intermediates. The resulting time constants are, however, in good agreement with those from step-scan spectroscopy (Figure S2). The worse statistics of the fit is attributed to the much smaller spectral range included in the analysis. (B) The SADS resulting from the global fit show the typical features at around 1189 (+) cm -1 and 1200 (-) cm -1 of the four intermediates K L, L, M and N in this spectral range. The K L intermediate is not included in the simplified photocycle presented in Fig. 3b, because it is silent in the visible spectral range, but has been analyzed in detail by infrared spectroscopy. 1,2 S2

3 Figure S2: Global fit of results from step-scan FTIR spectroscopy. (A) The concentration profiles were obtained from the same procedure as for dual-comb spectroscopy (Figure S1). The fitted concentration profiles (straight lines) agree well with the experimental data (points). (B) SADS of the intermediates K L, L, M and N from the global fit of the data set from step-scan FTIR spectroscopy. The spectral range of the dual-comb spectrometer is highlighted in grey. Much more elaborate kinetic models have been applied to the bacteriorhodopsin photocycle previously, 3,4 but the simplified model used here reproduces the known SADS of the respective intermediates and is, therefore, considered to be sufficient for a comparison to the results from dual-comb spectroscopy. S3

4 Figure S3: Representativeness of a single shot. Single-shot spectrum at 8 cm -1 for the time range of µs in comparison to the average over 50 shots with the standard deviation shown as error bars. Figure S4: Influence of probe light exposure on the samples. Bacteriorhodopsin samples were exposed for different durations to the QCL emission with a power of 82 mw. The kinetics averaged over 100 shots and 10 cm -1 and are highly similar and do not indicate any degradation by the probe light. S4

5 Figure S5: Infrared absorption spectra of bacteriorhodopsin samples with different path lengths. Representative absorption spectra of the samples used for the analysis by step-scan spectroscopy and dualcomb spectroscopy. The path lengths of about 4 µm and 16 µm were estimated from the absorbance of the water libration mode at 2130 cm -1. The high ratio of absorbance of amide I/water at 1650 cm -1 to amide II at 1550 cm -1 provides evidence of a well hydrated sample. S5

6 Figure S6: Comparison of dual-comb experiments on samples with different path lengths. (A) The kinetics of samples with different path lengths (Figure S5) are compared at selected wavelengths. After scaling to the maximal difference absorbance of 1200 cm -1, the kinetics become highly similar irrespective of an increase in path length by a factor of about four. (B) The comparison of the kinetics of two samples with different path lengths without any scaling shows an increase in difference signal achieved by the increase in path length. Moreover, such an increase facilitates strongly the application of flow systems in H 2 O preventing any clogging 5 or high pressure. 6 S6

7 Figure S7: Global fit of results from dual-comb spectroscopy at different ph values. Concentration profiles at ph 6.0 (A) and at ph 9.0 (B) show a slower formation of N at higher ph with time constants of 2.9 and 3.8 ms, respectively, and a slower decay of N with time constants of 6.8 ms and >18 ms, respectively. S7

8 Figure S8: Global fit of a single shot from dual-comb spectroscopy and standard deviations. Resulting concentration profiles at ph 6.0 are shown. Global fits of 10 consecutive single shots yielded mean time constants with standard deviations of τ 2 = (105 ± 64) µs, τ 3 = (2.2 ± 0.9) ms and τ 4 = (8.3 ± 2.1) ms. The extracted time constants are consistent with those analyzed after averaging 500 shots albeit lacking τ 1 in the early microsecond range (Figure S7A). Much better results from global fitting would be obtained at the single-shot level, if the accessible spectral range was expanded. Supporting References (1) Yuzawa, T.; Kato, C.; George, M. W.; Hamaguchi, H. O. Appl. Spectrosc. 1994, 48, (2) Braiman, M. S.; Bousche, O.; Rothschild, K. J. Proc. Natl. Acad. Sci. U. S. A. 1991, 88, (3) Chizhov, I.; Chernavskii, D. S.; Engelhard, M.; Mueller, K. H.; Zubov, B. V.; Hess, B. Biophys. J. 1996, 71, (4) Lozier, R. H.; Bogomolni, R. A.; Stoeckenius, W. Biophys. J. 1975, 15, (5) Brandstetter, M.; Volgger, L.; Genner, A.; Jungbauer, C.; Lendl, B. Appl. Phys. B 2013, 110, (6) Thöing, C.; Pfeifer, A.; Kakorin, S.; Kottke, T. Phys. Chem. Chem. Phys. 2013, 15, S8