Spectroscopic reaction monitoring is a proven tool in R&D and production environments, where it is used to gain insight into chemical reactions. In this presentation we showcase how dual-comb spectroscopy (DCS) can be used to monitor protein dynamics on a microsecond to second time-scale. Kinetics of irreversible protein reactions require an analytical technique that provides time-dependent infrared spectra in a single shot. First results have been validated with the recording of transients of the photoactivated proton pump bacteriorhodopsin with DCS and stepScan FTIR [1].
FTIR with time resolutions faster than 10 ms is only possible with photoactivatable proteins, where the reactions can be repeated hundreds of times because they undergo a photocycle. Many relevant protein reactions are non-repetitive. In many cases reactions can be induced by caged compounds [2]. As an example, we investigate the inhibiting Gi protein and the larger protein-protein complex of Gai with its cognate regulator of G-protein signaling (RGS). We compare caged compound induced reactions monitored by FTIR and DCS. With DCS we observe good data quality with 4 µs time resolution, three orders of magnitude faster than any FTIR measurement. DCS allows for infrared spectroscopic studies in the so-far unresolvable microsecond time regime for non-repetitive biological systems including GTPases and ATPases.
Additionally, we will present how dual-comb spectroscopy can be used to monitor fast kinetic reactions in combination with the stopped-flow technique, which was previously used more with UV-VIS or fluoresce because of lack of speed of currently available FTIR spectrometers. Mid-IR spectroscopy is however much more sensitive and specific compared to these other techniques and will pave the way to new research possibilities for kinetic studies.
[1] J. L. Klocke, M. Mangold, P. Allmendinger, A. Hugi, M. Geiser, P. Jouy, J. Faist, T. Kottke, (2018) Anal. Chem. 90, 17, 10494–10500. “Single-Shot Sub-microsecond Mid-infrared Spectroscopy on Protein Reactions with Quantum Cascade Laser Frequency Combs“
[2] M. J. Norahan, R. Horvath, N. Woitzik, P. Jouy, F. Eigenmann, K. Gerwert, C. Kötting (2021) Anal. Chem. 2021, 93, 17, 6779–6783 „Microsecond resolved infrared spectroscopy on non-repetitive protein reactions by applying caged-compounds and quantum cascade laser frequency combs“
Speaker
Florian Eigenmann
Florian holds a Ph.D. in Chemistry from ETH Zurich and joined IRsweep in 2018 as Head of Sales. He was instrumental in releasing the high spectral resolution version of the IRis-F1 and in developing reaction monitoring applications including stopped flow and curing monitoring. Previously, he was with Bruker Optics and Mettler Toledo for a total of 12 years in Country Manager and Business Development positions for mid-IR applications with a focus on process analytics.