Confinement in crystal lattice alters entire photocycle pathway of the Photoactive Yellow Protein

Abstract

Femtosecond time-resolved crystallography (TRC) on proteins enables resolving the spatial structure of short-lived photocycle intermediates. An open question is whether confinement and lower hydration of the proteins in the crystalline state affect the light-induced structural transformations. Here, we measured the full photocycle dynamics of a signal transduction protein often used as model system in TRC, Photoactive Yellow Protein (PYP), in the crystalline state and compared those to the dynamics in solution, utilizing electronic and vibrational transient absorption measurements from 100 fs over 12 decades in time. We find that the photocycle kinetics and structural dynamics of PYP in the crystalline form deviate from those in solution from the very first steps following photon absorption. This illustrates that ultrafast TRC results cannot be uncritically extrapolated to in vivo function, and that comparative spectroscopic experiments on proteins in crystalline and solution states can help identify structural intermediates under native conditions.

QM calculations predict the energetics and infrared spectra of transient glutamine isomers in LOV photoreceptors

“…Time-resolved infrared spectra of EL222 (17-225) were obtained at time delays ranging from femtoseconds to submilliseconds and were performed on a setup described before. 28,[79][80][81] The pump wavelength was tuned to 475 nm while the probe beam covered the mid-IR range from 1511 to 1759 cm À1 with 4 cm À1 spectral resolution. 108 time points were acquired from À100 ns to 702 ms with background spectra measured at negative time delays.…”

The molecular pH-response mechanism of the plant light-stress sensor PsbS

“…Two-dimensional infrared spectroscopy (2DIR) was carried out in the pump-probe geometry as previously described 61,62 , as an extension to an existing femtosecond mid-IR spectrometer 46,63,64 . In short, femtosecond mid-IR pulses were generated via difference frequency generation in Ag 2 GaS 2 (100 fs pulse duration, 200 cm −1 bandwidth) pumped by a Ti:sapphire regenerative amplifier with 1 kHz repetition rate.…”

Hydration and its Hydrogen Bonding State on a Protein Surface in the Crystalline State as Revealed by Molecular Dynamics Simulation

“…In this MD simulation in solution, the solvent density and solvent dipole on the protein surface were in good agreement with the hydration structure observed by crystal structure analysis, indicating that the combination of computational and experimental analysis is effective in protein hydration studies. Most MD simulations of proteins are performed in solution, but the protein’s conformation, dynamics, and function are changed in the crystalline state from in a solution ( Li et al, 2017 ; Srivastava et al, 2018 ; Konold et al, 2020 ). Because the limitation of crystallography in not analyzing the protein dynamics is discussed ( Srivastava et al, 2018 ), MD simulations in a protein crystalline state should be useful to compare them to experimental protein crystallography.…”