Microsecond-Resolved Infrared Spectroscopy on Nonrepetitive Protein Reactions by Applying Caged Compounds and Quantum Cascade Laser Frequency Combs

Abstract

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 nonrepetitive reactions, e.g., induced by caged compounds, were limited to the millisecond time domain. The advent of dual-comb quantum cascade laser now allows for a rapid reaction monitoring in the microsecond 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 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 a 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 microsecond time regime for nonrepetitive biological systems including all GTPases and ATPases.

A mid-infrared lab-on-a-chip for dynamic reaction monitoring

“…(i) Dynamic processes, such as those found in chemical reactions 24 or conformational changes, i.e., structural changes of a molecules three-dimensional structure 13 , reveal important characteristics that have to be analyzed with high temporal resolution for their adequate investigation. An in situ sensor for label-free real-time measurements is the ideal tool for monitoring those analyte-changes, completely avoiding time-consuming offline analytics.…”

Time-Resolved Vibrational and Electronic Spectroscopy for Understanding How Charges Drive Metal Oxide Catalysts for Water Oxidation

“…For advancing the temporal resolution and sensitivity of vibrational spectroscopy in order to access every surface intermediate and elucidating its true kinetic behavior, new spectroscopic tools and new techniques for triggering heterogeneous catalysis are emerging. A new broadband infrared probing technique with improved sensitivity is dual-comb quantum cascade laser spectroscopy that affords hundreds of nanoseconds time resolution in the mid-infrared region (pulsed pump–continuous probe spectroscopy). , For fast monitoring of (dark) electrocatalytic water oxidation mechanisms with this infrared technique, triggering of reaction needs to be conducted with nanosecond voltage jumps. This should be feasible by directly interfacing the electrocatalyst layer with a photonic semiconductor, e.g., GaP film.…”

Mid-infrared dual-comb polarimetry of anisotropic samples

“…The operating principles of dual-comb spectroscopy have been described previously [33][34][35] . Briefly, FC 1 and FC 2 deliver frequency combs with inter-line spacings (f rep,1 and f rep,2 ) of about 10 GHz.…”