Serial femtosecond crystallography using ultrashort pulses from X-ray Free Electron Lasers (XFELs) offers the possibility to study light-triggered dynamics of biomolecules. Using microcrystals of the blue light photoreceptor, photoactive yellow protein, as a model system, we present high resolution, time-resolved difference electron density maps of excellent quality with strong features, which allow the determination of structures of reaction intermediates to 1.6 Å resolution. These results open the way to the study of reversible and non-reversible biological reactions on time scales as short as femtoseconds under conditions which maximize the extent of reaction initiation throughout the crystal.
A variety of organisms have evolved mechanisms to detect and respond to light, in which the response is mediated by protein structural changes following photon absorption. The initial step is often the photo-isomerization of a conjugated chromophore. Isomerization occurs on ultrafast timescales, and is substantially influenced by the chromophore environment. Here we identify structural changes associated with the earliest steps in the trans to cis isomerization of the chromophore in photoactive yellow protein. Femtosecond, hard X-ray pulses emitted by the Linac Coherent Light Source were used to conduct time-resolved serial femtosecond crystallography on PYP microcrystals over the time range from 100 femtoseconds to 3 picoseconds to determine the structural dynamics of the photoisomerization reaction.
Wild type green fluorescent protein (GFP) from Aequorea victoria absorbs predominantly at 398 nm. Illumination with UV (254 nm) or visible (390 nm) light transforms this state (GFP(398)) into one absorbing at 483 nm (GFP(483)). Here we show that this photoconversion of GFP is a one-photon process that is paralleled by decarboxylation of Glu 222. We propose a mechanism in which decarboxylation is due to electron transfer between the gamma-carboxylate of Glu 222 and the p-hydroxybenzylidene-imidazolidinone chromophore of GFP, followed by reverse transfer of an electron and a proton to the remaining carbon side chain atom of Glu 222. Oxidative decarboxylation of a gamma-carboxylate represents a new type of posttranslational modification that may also occur in enzymes with high-potential reaction intermediates.
The fluorescence properties of GFP are strongly influenced by the protonation states of its chromophore and nearby amino acid side chains. In the ground state, the GFP chromophore is neutral and absorbs in the near UV. Upon excitation, the chromophore is deprotonated, and the resulting anionic chromophore emits its green fluorescence. So far, only excited-state intermediates have been observed in the GFP photocycle. We have used ultrafast multipulse control spectroscopy to prepare and directly observe GFP's hidden anionic ground-state intermediates as an integral part of the photocycle. Combined with dispersed multichannel detection and advanced global analysis techniques, the existence of two distinct anionic ground-state intermediates, I 1 and I2, has been unveiled. I1 and I2 absorb at 500 and 497 nm, respectively, and interconvert on a picosecond timescale. The I 2 intermediate has a lifetime of 400 ps, corresponding to a proton back-transfer process that regenerates the neutral ground state. Hydrogen͞deuterium exchange of the protein leads to a significant increase of the I1 and I2 lifetimes, indicating that proton motion underlies their dynamics. We thus have assessed the complete chain of reaction intermediates and associated timescales that constitute the photocycle of GFP. Many elementary processes in biology rely on proton transfers that are limited by slow diffusional events, which seriously precludes their characterization. We have resolved the true reaction rate of a proton transfer in the molecular ground state of GFP, and our results may thus aid in the development of a generic understanding of proton transfer in biology.hidden reaction intermediates ͉ multipulse control spectroscopy ͉ proton transfer ͉ ultrafast spectroscopy
The P(r) to P(fr) transition of recombinant Synechocystis PCC 6803 phytochrome Cph1 and its N-terminal sensor domain Cph1Delta2 is accompanied by net acidification in unbuffered solution. The extent of this net photoreversible proton release was measured with a conventional pH electrode and increased from less than 0.1 proton released per P(fr) formed at pH 9 to between 0.6 (Cph1) and 1.1 (Cph1Delta2) H(+)/P(fr) at pH 6. The kinetics of the proton release were monitored at pH 7 and pH 8 using flash-induced transient absorption measurements with the pH indicator dye fluorescein. Proton release occurs with time constants of approximately 4 and approximately 20 ms that were also observed in parallel measurements of the photocycle (tau(3) and tau(4)). The number of transiently released protons per P(fr) formed is about one. This H(+) release phase is followed by a proton uptake phase of a smaller amplitude that has a time constant of approximately 270 ms (tau(5)) and is synchronous with the formation of P(fr). The acidification observed in the P(r) to P(fr) transition with pH electrodes is the net effect of these two sequential protonation changes. Flash-induced transient absorption measurements were carried out with Cph1 and Cph1Delta2 at pH 7 and pH 8. Global analysis indicated the presence of five kinetic components (tau(1)-tau(5): 5 and 300 micros and 3, 30, and 300 ms). Whereas the time constants were approximately pH independent, the corresponding amplitude spectra (B(1), B(3), and B(5)) showed significant pH dependence. Measurements of the P(r)/P(fr) photoequilibrium indicated that it is pH independent in the range of 6.5-9.0. Analysis of the pH dependence of the absorption spectra from 6.5 to 9.0 suggested that the phycocyanobilin chromophore deprotonates at alkaline pH in both P(r) and P(fr) with an approximate pK(a) of 9.5. The protonation state of the chromophore at neutral pH is therefore the same in both P(r) and P(fr). The light-induced deprotonation and reprotonation of Cph1 at neutral pH are thus due to pK(a) changes in the protein moiety, which are linked to conformational transitions occurring around 4 and 270 ms after photoexcitation. These transient structural changes may be relevant for signal transduction by this cyanobacterial phytochrome.
The photoreactions of the Pr ground state of cyanobacterial phytochrome Cph1 from Synechocystis PCC 6803 have been investigated by picosecond time-resolved mid-infrared spectroscopy at ambient temperature. With femtosecond excitation of the Pr state at 640 nm, the photoisomerized Lumi-R product state is generated with kinetics and associated difference spectra indicative of vibrational cooling with tau(1) = 3 ps time constant and excited state decay with tau(1) = 3 ps, tau(2) = 14 ps, and tau(3) = 134 ps time constants. The Lumi-R state is characterized by downshifted absorption of three C=C modes assigned to C(15)=C(16), C(4)=C(6), and a delocalized C=C mode, in addition to the downshifted C(19)=O mode. The Lumi-R minus Pr difference spectrum is indicative of global restructuring of the chromophore on the ultrafast timescale, which is discussed in light of C(15) Z/E photoisomerization in addition to changes near C(5), which could be low bond order torsional angle changes.
The reversible photoswitching between the 'on' and 'off' states of the fluorescent protein Dronpa involves photoisomerization as well as protein side-chain rearrangements, but the process of interconversion remains poorly characterized. Here we use time-resolved infrared measurements to monitor the sequence of these structural changes, but also of proton transfer events, which are crucial to the development of spectroscopic contrast. Light-induced deprotonation of the chromophore phenolic oxygen in the off state is a thermal ground-state process, which follows ultrafast (9 ps) trans-cis photoisomerization, and so does not involve excited-state proton transfer. Steady-state infrared difference measurements exclude protonation of the imidazolinone nitrogen in both the on and off states. Pump-probe infrared measurements of the on state reveal a weakening of the hydrogen bonding between Arg66 and the chromophore C ¼ O, which could be central to initiating structural rearrangement of Arg66 and His193 coinciding with the low quantum yield cis-trans photoisomerization
J.J.v.T. is a Royal Society University Research Fellow. We thank Gunnar Jeschke for help with initial experiments, for help with DEER data analysis and for valuable discussions. We thank Peter Höfer for access to facilities at Bruker BioSpin, Rheinstetten. We thank Jim McDonnell for help with acquiring NMR data. We acknowledge access to beamline ID09B at the European Synchrotron Radiation Facility under access number SC2438. We thank Michael Wulff for support at beamline ID09B at ESRF. We thank Jungkweon Choi for help with collecting TR-WAXS data. We thank Alexander Grishaev for help with the NIH CNS-SAXS program. J.J.v. Supporting information:Nuclear magnetic resonance, double electron electron resonance, and small angle X-ray scattering tables; figures; and experimental details. This material is available free of charge via the Internet at http://pubs.acs.org reduced folding of the I 2 ′ state (also referred to as the 'pB' state) that is widely reported, but indicates it to be relatively ordered and rearranged. Furthermore, there is direct evidence for the repositioning of the N-terminal region in the I 2 ′ state, which is structurally modeled by dynamical annealing and refinement calculations.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
334 Leonard St
Brooklyn, NY 11211
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.