Application: Photochemistry and photocatalysis, Protein dynamics, Time resolved vibrational spectroscopy
Product: IRis-F1
Luiz Schubert, Pit Langner, David Ehrenberg, Victor A. Lorenz-Fonfria, and Joachim Heberle
The Journal of Chemical Physics; 2022
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Mid-IR spectroscopy is a powerful and label-free technique to investigate protein reactions. In this study, we use quantum-cascade-laser-based dual-comb spectroscopy to probe protein conformational changes and protonation events by a single-shot experiment. By using a well-characterized membrane protein, bacteriorhodopsin, we provide a comparison between dual-comb spectroscopy and our homebuilt tunable quantum cascade laser (QCL)-based scanning spectrometer as tools to monitor irreversible reactions with high time resolution. In conclusion, QCL-based infrared spectroscopy is demonstrated to be feasible for tracing functionally relevant protein structural changes and proton translocations by single-shot experiments. Thus, we envisage a bright future for applications of this technology for monitoring the kinetics of irreversible reactions as in (bio-)chemical transformations.
Application: Photochemistry and photocatalysis, Protein dynamics
Product: IRis-F1
Klocke, Jessica L.; Mangold, Markus; Allmendinger, Pitt; Hugi, Andreas; Geiser, Markus; Jouy, Pierre; Faist, Jérôme; Kottke, Tilman
Analytical Chemistry, American Chemical Society; 2018
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The kinetic analysis of irreversible protein reactions requires an analytical technique that provides access to time-dependent infrared spectra in a single shot. Here, we present a spectrometer based on dual-frequency-comb spectroscopy using mid-infrared frequency combs generated by quantum cascade lasers. Attenuation of the intensity of the combs by molecular vibrational resonances results in absorption spectra covering 55 cm–1 in the fingerprint region. The setup has a native resolution of 0.3 cm–1, noise levels in the μOD range, and achieves sub-microsecond time resolution. We demonstrate the simultaneous recording of both spectra and transients of the photoactivated proton pump bacteriorhodopsin. More importantly, a single shot, i.e., a single visible light excitation, is sufficient to extract spectral and kinetic characteristics of several intermediates in the bacteriorhodopsin photocycle. This development paves the way for the noninvasive analysis of enzymatic conversions with high time resolution, broad spectral coverage, and minimal sample consumption.
Application: Protein dynamics
Product: IRis-F1
Schubert, Luiz
Presentation at ECSBM 2019; 2019
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Application: Photochemistry and photocatalysis, Protein dynamics, Time resolved vibrational spectroscopy
Product: IRis-F1
Geiser, Markus; Klocke, Jessica L.; Mangold, Markus; Allmendinger, Pitt; Hugi, Andreas; Jouy, Pierre; Horvath, Balint; Faist, Jerome; Kottke, Tilman
Biophysical Journal, Elsevier; 2018
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Time-resolved vibrational spectroscopy is an important tool for understanding biological processes and chemical reaction pathways. Today, all available methods to our knowledge require many repetitions of an experiment to acquire a microsecond time-res. mid-IR spectrum. We present the IRspectrometer, a quantum cascade laser dual frequency comb spectrometer. It allows for parallel acquisition of hundreds of mid-infrared wavelengths with microsecond time resolution. The formation of the light-activated L, M and N-states in bacteriorhodopsin - which only have µs to ms lifetimes - has been recorded with our setup: e.g. infrared response of bacteriorhodopsin to 10 ns visible light pulses with microsecond time-resolution. The different wavelengths were all measured in parallel thanks to the dual-comb approach. The spectra as well as the kinetics show good agreement with those from step-scan FTIR measurements. As a benchmark, the spectral signature of several intermediate states of the bacteriorhodopsin photocycle has been recorded in a single shot measurement. This approach greatly reduces the complexity of time-resolved bio-spectroscopy measurements in the mid-infrared which currently require many repetitions.
Application: Protein dynamics
Product: IRis-F1
Dutton, Gail
Genetic Engineering & Biotechnology News; 2018
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Application: Photochemistry and photocatalysis, Protein dynamics, Time resolved vibrational spectroscopy
Product: IRis-F1
Mohamad Javad Norahan, Raphael Horvath, Nathalie Woitzik, Pierre Jouy, Florian Eigenmann, Klaus Gerwert and Carsten Koetting
ACS Analytical Chemistry; 2021
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Infrared spectroscopy is ideally suited for the investigation of protein reactions at the atomic level. Many systems were investigated successfully by applying Fourier transform infrared (FTIR) spectroscopy. While rapid-scan FTIR spectroscopy is limited by time resolution (about 10 ms with 16 cm-1 resolution), step-scan FTIR spectroscopy reaches a time-resolution of about 10 ns but is limited to cyclic reactions that can be repeated hundreds of times under identical conditions. Consequently, FTIR with high time resolution was only possible with photoactivable proteins that undergo a photocycle. The huge number of non-repetitive reactions, e.g. induced by caged compounds, were limited to the ms time domain. The advent of dual comb quantum cascade laser allows now for a rapid reaction monitoring in the μs time domain. Here we investigate the potential to apply such an instrument to the huge class of G-proteins. We compare caged-compound induced reactions monitored by FTIR and dual comb spectroscopy, respectively, by applying the new technique to the α subunit of the inhibiting Gi protein and to the larger protein-protein complex of Gαi with its cognate regulator of G-protein signaling (RGS). We observe good data quality with 4 μs time resolution with a wavelength resolution comparable to FTIR. This is more than three orders of magnitude faster than any FTIR measurement on G-proteins in the literature. This study paves the way for infrared spectroscopic studies in the so far unresolvable μs time regime for non-repetitive biological systems including all GTPases and ATPases.
Application: Protein dynamics, Stopped flow and microfluidics
Product: IRis-F1
Markus Geiser, Raphael Horvath and Jakob Hayden
Photonics Views; 2021
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Dual‐comb spectroscopy is a powerful direct absorption spectroscopy technique and has attracted considerable attention, with high precision spectroscopy applications being the most prominent since the invention of the frequency comb was awarded the 2005 Nobel Prize in physics. The application range is continuously broadening and here we review recent advances achieved with quantum cascade laser frequency comb spectrometers in process monitoring and reaction kinetics applications.