The Short-Lived Signaling State of the Photoactive Yellow Protein Photoreceptor Revealed by Combined Structural Probes

Abstract

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.

Protein dynamics and function from solution state NMR spectroscopy

“…trSAXS was used to describe global conformational changes on a time scale of seconds while R 2 -relaxation dispersion experiments were used to characterize dynamics on the millisecond time scale. The potential of combining SAXS with NMR (along with a range of other techniques) is also demonstrated by the work of the van Thor group on the photoactive yellow protein (Ramachandran et al 2011).…”

“…In one notable example, DEER experiments using spin-labeled frozen samples (T = 60 K) of the photoactive yellow protein showed that the distance distribution of the labels changed on exposure of the photoreceptor to light (Ramachandran et al 2011). In the same study, timeresolved pump-probe x-ray scattering (TR-SAXS/WAXS) (Cammarata et al 2008) was used to demonstrate that the radius of gyration and particle size both increased when the protein was exposed to light (Ramachandran et al 2011). Chemical shift perturbations and classical NOE data were combined with TR-SAXS/WAXS and DEER data to produce a model of the protein's dynamic light-exposed state (Ramachandran et al 2011).…”

“…In the same study, timeresolved pump-probe x-ray scattering (TR-SAXS/WAXS) (Cammarata et al 2008) was used to demonstrate that the radius of gyration and particle size both increased when the protein was exposed to light (Ramachandran et al 2011). Chemical shift perturbations and classical NOE data were combined with TR-SAXS/WAXS and DEER data to produce a model of the protein's dynamic light-exposed state (Ramachandran et al 2011).…”

Synchrotron ultrafast techniques for photoactive transition metal complexes

“…The myoglobin tertiary structure relaxation in response to the optically induced CO dissociation [172,183] and refolding of cytochrome c [172] were also investigated. Using a combination of double electron-electron resonance spectroscopy, NMR and TR-SAXS/WAXS, Ramachandran et al first succeeded in directly monitoring the structural evolution of the transient signalling state of the photoactive yellow protein, considered a prototype photoreceptor [184]. These results were lately complemented by a detailed TR-SAXS/WAXS investigation of the structural changes occurring during the photocycle in a wide time range from 3.16 µs to 300 ms [185].…”

Tryptophan fluorescence as a reporter for structural changes in photoactive yellow protein elicited by photo-activation

“…30 From a study with fluorescein labelled PYP in the N-terminal cap it is known that the structural changes in the N-terminus start in the regions around residue 5 upon formation of pB′ and they are propagated in pB to the rest of the N-terminal cap. 71 In the pB structure determined with DEER spectroscopy, NMR, and TR-SAXS/ WAXS, the first 7 N-terminal residues are separated from the rest of the protein and completely exposed to the solvent, 72 whereas in the ground state residue 6 is close to the β-sheet and the N-terminus is more compact. 13 This is in line with the large red shift we observe for W6 upon the formation of pB.…”