Microsecond resolved infrared spectroscopy on non-repetitive 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 (about10 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.

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

“…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). 62,63 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.…”

Time-resolved infrared difference spectroscopy in cells: Response of the basic region leucine zipper of aureochrome

“…Moreover, ICIRD spectroscopy is currently limited to slight overexpression with a protein concentration between 120 μM and 330 µM [28] deviating from a native receptor concentration. Further improvements in time resolution and sensitivity might be achieved by employing setups with quantum cascade lasers (QCL) [58][59][60] or QCL frequency combs [53,61]. Such approaches might allow in future in-cell infrared spectroscopic studies at native receptor concentration and nanosecond time resolution.…”

“…Here, we established the time-resolved ICIRD spectroscopy on LOV1-C57S-LOV2 with a time resolution of 7.6 ms at a spectral resolution of 4 cm −1 . For comparison, the time resolution of commercially available rapid-scan FTIR spectroscopy was estimated to be about 10 ms at a spectral resolution of only 16 cm −1 [53], whereas home-built rapid-scan setups reach 13 μs at a spectral resolution of 9 cm −1 [54]. To achieve the high time resolution with a conventional FTIR spectrometer, we delayed the TTL signal from the spectrometer to the pulsed laser to compensate for the void time of the interferometer during the mirror inversion.…”

Coherently-Averaged Dual-Comb Spectrometer at 7.7 µm with Master and Follower Quantum Cascade Lasers

“…Mid-infrared (MIR) quantum cascade laser (QCL) frequency combs [2] are an appealing and thriving technology for DCS [3,4], as they cover strong spectroscopic absorption bands of many relevant molecules in the MIR region. Dual QCL comb spectrometers have become commercially available as a table-top instrument, which is leading to new discoveries in many fields of science including biology and chemistry [5][6][7][8][9][10][11][12]. One key advantage of QCL-based DCS is the ability to perform studies with up to sub-microsecond time-resolution [11].…”