Serial Femtosecond Crystallography Reveals that Photoactivation in a Fluorescent Protein Proceeds via the Hula Twist Mechanism

Abstract

Chromophore cis/trans photoisomerization is a fundamental process in chemistry and in the activation of many photosensitive proteins. A major task is understanding the effect of the protein environment on the efficiency and direction of this reaction compared to what is observed in the gas and solution phases. In this study, we set out to visualize the hula twist (HT) mechanism in a fluorescent protein, which is hypothesized to be the preferred mechanism in a spatially constrained binding pocket. We use a chlorine substituent to break the twofold symmetry of the embedded phenolic group of the chromophore and unambiguously identify the HT primary photoproduct. Through serial femtosecond crystallography, we then track the photoreaction from femtoseconds to the microsecond regime. We observe signals for the photoisomerization of the chromophore as early as 300 fs, obtaining the first experimental structural evidence of the HT mechanism in a protein on its femtosecond-to-picosecond timescale. We are then able to follow how chromophore isomerization and twisting lead to secondary structure rearrangements of the protein β-barrel across the time window of our measurements.

Structural Characterization of Fluorescent Proteins Using Tunable Femtosecond Stimulated Raman Spectroscopy

“…In fact, the way of preparing the crystal sometimes can alter the photophysical and photochemical properties of an FP. In a recent work on the cis-to-trans photoisomerization pathways of photoswitchable FPs, Chang et al investigated a reversibly switchable enhanced GFP (rsEGFP2) variant containing a monochlorinated Ala-Tyr-Gly (AYG) chromophore, where the resultant structural symmetry breaking enables the differentiation between one-bondflip and hula-twist mechanisms via X-ray crystallography [16,17]. The authors found that the exact pathway depends on the packing of the protein, which is experimentally controllable, that is, the more volume-demanding one-bond-flip pathway is favored in an expanded crystal lattice, while the hula-twist pathway prevails in a tighter packing configuration.…”

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 .…”

XFEL Beamline Optical Instrumentation for Ultrafast Science

“…Since the first demonstrations of femtosecond time-resolved pump–probe protein X-ray crystallography on Myoglobin in 2015 and the PYP in 2016, quite a number of additional examples have been shown. ,,, Until recently, most of these studies have been interpreted essentially using rate kinetics arguments. It is an open question of under which conditions such methods are applicable on the ultrafast time scale.…”

“…Diffractive techniques in the pump–probe geometry allow for direct recording of structural changes in materials and have been highly successful at XFELs. Time-resolved serial femtosecond crystallography (TR-SFX) has captured numerous structural molecular movies of biologically relevant targets including photoactive yellow protein (PYP), − photoswitching derivatives of green fluorescent protein, − rhodopsin, , photosystem II, , ribonucleotide reductase, myoglobin, and photolyase. , TR-SFX has the caveat of requiring crystalline samples. At FELs, it is performed in a serial manner, where a new crystal is introduced for each new X-ray shot.…”