Needles in a haystack: H-bonding in an optogenetic protein observed with isotope labeling and 2D-IR spectroscopy

Abstract

Recently, re-purposing of cyanobacterial photoreceptors as optogentic actuators enabled light-regulated protein expression in different host systems. These new bi-stable optogenetic tools enable interesting new applications, but their light-driven working mechanism...

Vibrational couplings between protein and cofactor in bacterial phytochrome Agp1 revealed by 2D-IR spectroscopy

“…2 C and D ). CO D thus acts as a local vibrational probe inside the protein environment and reports on the changes of its immediate surroundings, such as hydrogen bond dynamics ( 35 ). First, CO D is downshifted in Pfr by about 2 cm 1 in good agreement with previously published linear IR data ( 36 , 37 ).…”

“…Agp1 (PAS-GAF-PHY) samples were prepared according to published protocols in a Tris buffer containing 50 mM Tris-Cl, 5 mM EDTA, and 300 mM NaCl and adjusted with HCl and NaOH to pH 7.8 ( 48 ). The 2D-IR spectra were collected with a setup based on a 100-kHz Yb-fiber laser, mid-IR optical parametric amplifier (OPA), and a pulse shaper as described before in detail ( 35 , 46 ). Here, the 2D-IR spectra were collected in the rotating frame with a reference frequency of 1,450 cm 1 , while the step size for scanning the t 1 delay was 40 fs, and the t 2 delay was 200 fs for all measurements.…”

Red Light Optogenetics in Neuroscience

“…New knowledge has been gained on the structure of red light-sensing opsins ( Oda et al, 2018 ) and the activation dynamics of CBCRs ( Wang D. et al, 2020 ; Ruf and Buhrke, 2021 ). Novel advances with phytochromes include the structural characterization of the plant phytochrome ( Tahir ul Qamar et al, 2021 ) and increased knowledge about signal transduction in the bacterial phytochromes ( Böhm et al, 2021 ).…”

The impact of chromophore choice on the assembly kinetics and primary photochemistry of a red/green cyanobacteriochrome

“…The apo-and holo-proteins were expressed and purified as described previously. 17 In vitro assembly experiments 2 mL of 3 µM bilin in aqueous buffer were prepared in a 1 cm pathlength quartz cuvette and continuously stirred throughout the experiment. The buffer was based on standard PBS with additional 5 mM EDTA to scavenge metal ions and 5 mM β-mercaptoethanol as reducing agent to keep the canonical Cys thiol in a reduced state.…”