Optical control of ultrafast structural dynamics in a fluorescent protein

Abstract

The photoisomerization reaction of a fluorescent protein chromophore occurs on the ultrafast timescale. The structural dynamics that result from femtosecond optical excitation have contributions from vibrational and electronic processes and from reaction dynamics that involve the crossing through a conical intersection. The creation and progression of the ultrafast structural dynamics strongly depends on optical and molecular parameters. When using X-ray crystallography as a probe of ultrafast dynamics, the origin of the observed nuclear motions is not known. Now, high-resolution pump–probe X-ray crystallography reveals complex sub-ångström, ultrafast motions and hydrogen-bonding rearrangements in the active site of a fluorescent protein. However, we demonstrate that the measured motions are not part of the photoisomerization reaction but instead arise from impulsively driven coherent vibrational processes in the electronic ground state. A coherent-control experiment using a two-colour and two-pulse optical excitation strongly amplifies the X-ray crystallographic difference density, while it fully depletes the photoisomerization process. A coherent control mechanism was tested and confirmed the wave packets assignment.

Influence of pump laser fluence on ultrafast myoglobin structural dynamics

“…In time-resolved pump–probe SFX experiments, microcrystals are delivered into the XFEL beam using mostly liquid jets and diffraction data are collected at distinct time delays following a photo-exciting pump laser flash. Using femtosecond- to sub-picosecond-long laser flashes, this approach has been used to study isomerization reactions in photoactive yellow protein 4 , fluorescent proteins 5 , 16 , 17 , various rhodopsins 6 , 7 , 9 , 10 , 12 , 14 and phytochrome 8 ; electron transfer reactions in a photosynthetic reaction centre 11 and photolyase 13 , 22 , 23 ; photodecarboxylation 24 and photodissociation 3 . In all cases, a very high pump laser fluence was used to maximize the light-induced difference electron density signal 20 .…”

“…On timescales ranging from femtoseconds to milliseconds and for a variety of biological systems, time-resolved serial femtosecond crystallography (TR-SFX) has provided detailed structural data for light-induced isomerization, breakage or formation of chemical bonds and electron transfer 1 , 2 . However, all ultrafast TR-SFX studies to date have employed such high pump laser energies that nominally several photons were absorbed per chromophore 3 – 17 . As multiphoton absorption may force the protein response into non-physiological pathways, it is of great concern 18 , 19 whether this experimental approach 20 allows valid conclusions to be drawn vis-à-vis biologically relevant single-photon-induced reactions 18 , 19 .…”

Kilohertz droplet-on-demand serial femtosecond crystallography at the European XFEL station FXE

“…X-ray Free Electron Lasers (XFELs) have proven to be a powerful tool for studying chemical and biological mechanisms through crystallography. 1–7 XFELs deliver femtosecond pulses with peak brilliance over a billion times more intense than that of their synchrotron counterparts. 8 The short duration of the pulses allows tracking of structural and coherent dynamics on a sub-picosecond level, 1,9 for many photoreactions where the initial product-determining reaction step occurs.…”

“… 1–7 XFELs deliver femtosecond pulses with peak brilliance over a billion times more intense than that of their synchrotron counterparts. 8 The short duration of the pulses allows tracking of structural and coherent dynamics on a sub-picosecond level, 1,9 for many photoreactions where the initial product-determining reaction step occurs. However, due to the high peak brilliance, these X-rays destroy the sample upon exposure.…”

“… 25–28 Droplet-on-demand (DoD) injection methods have proved very successful at delivering high data rates with small sample consumption at ∼100 Hz FELs. 1,29–34 While the repetition rate of this method is physically capped by the acoustic wave generation and dampening speed of the injector, this technology can be theoretically be advanced to MHz operation. 35,36 The feasibility of this method at the latest high repetition rate FELs is yet to be demonstrated past 100 Hz.…”