Sensory proteins must relay structural signals from the sensory site over large distances to regulatory output domains. Phytochromes are a major family of red-light sensing kinases that control diverse cellular functions in plants, bacteria, and fungi.1-9 Bacterial phytochromes consist of a photosensory core and a C-terminal regulatory domain.10,11 Structures of photosensory cores are reported in the resting state12-18 and conformational responses to light activation have been proposed in the vicinity of the chromophore.19-23 However, the structure of the signalling state and the mechanism of downstream signal relay through the photosensory core remain elusive. Here, we report crystal and solution structures of the resting and active states of the photosensory core of the bacteriophytochrome from Deinococcus radiodurans. The structures reveal an open and closed form of the dimeric protein for the signalling and resting state, respectively. This nanometre scale rearrangement is controlled by refolding of an evolutionarily conserved “tongue”, which is in contact with the chromophore. The findings reveal an unusual mechanism where atomic scale conformational changes around the chromophore are first amplified into an Ångström scale distance change in the tongue, and further grow into a nanometre scale conformational signal. The structural mechanism is a blueprint for understanding how the sensor proteins connect to the cellular signalling network.

A ‘protein quake’ describes the hypothesis that proteins rapidly dissipate energy through quake like structural motions. Here we measure ultrafast structural changes in the Blastochloris viridis photosynthetic reaction center following multi-photon excitation using time-resolved wide angle X-ray scattering at an X-ray free electron laser. A global conformational change arises within picoseconds, which precedes the propagation of heat through the protein. This motion is damped within a hundred picoseconds.

Time-resolved x-ray solution scattering reveals the conformational signaling mechanism of a bacterial phytochrome.

Proteins serve as molecular machines in performing their biological functions, but the detailed structural transitions are difficult to observe in their native aqueous environments in real time. For example, despite extensive studies, the solution-phase structures of the intermediates along the allosteric pathways for the transitions between the relaxed (R) and tense (T) forms have been elusive. In this work, we employed picosecond X-ray solution scattering and novel structural analysis to track the details of the structural dynamics of wild-type homodimeric hemoglobin (HbI) from the clam Scapharca inaequivalvis and its F97Y mutant over a wide time range from 100 ps to 56.2 ms. From kinetic analysis of the measured time-resolved X-ray solution scattering data, we identified three structurally distinct intermediates (I1, I2, and I3) and their kinetic pathways common for both the wild type and the mutant. The data revealed that the singly liganded and unliganded forms of each intermediate share the same structure, providing direct evidence that the ligand photolysis of only a single subunit induces the same structural change as the complete photolysis of both subunits does. In addition, by applying novel structural analysis to the scattering data, we elucidated the detailed structural changes in the protein, including changes in the heme–heme distance, the quaternary rotation angle of subunits, and interfacial water gain/loss. The earliest, R-like I1 intermediate is generated within 100 ps and transforms to the R-like I2 intermediate with a time constant of 3.2 ± 0.2 ns. Subsequently, the late, T-like I3 intermediate is formed via subunit rotation, a decrease in the heme–heme distance, and substantial gain of interfacial water and exhibits ligation-dependent formation kinetics with time constants of 730 ± 120 ns for the fully photolyzed form and 5.6 ± 0.8 μs for the partially photolyzed form. For the mutant, the overall kinetics are accelerated, and the formation of the T-like I3 intermediate involves interfacial water loss (instead of water entry) and lacks the contraction of the heme–heme distance, thus underscoring the dramatic effect of the F97Y mutation. The ability to keep track of the detailed movements of the protein in aqueous solution in real time provides new insights into the protein structural dynamics.

BioCARS, a NIH-supported national user facility for macromolecular time-resolved X-ray crystallography at the Advanced Photon Source (APS), has recently completed commissioning of an upgraded undulator-based beamline optimized for single-shot laser-pump X-ray-probe measurements with time resolution as short as 100 ps. The source consists of two in-line undulators with periods of 23 and 27 mm that together provide high-flux pink-beam capability at 12 keV as well as first-harmonic coverage from 6.8 to 19 keV. A high-heat-load chopper reduces the average power load on downstream components, thereby preserving the surface figure of a Kirkpatrick-Baez mirror system capable of focusing the X-ray beam to a spot size of 90 µm horizontal by 20 µm vertical. A high-speed chopper isolates single X-ray pulses at 1 kHz in both hybrid and 24-bunch modes of the APS storage ring. In hybrid mode each isolated X-ray pulse delivers up to ~4 × 10(10) photons to the sample, thereby achieving a time-averaged flux approaching that of fourth-generation X-FEL sources. A new high-power picosecond laser system delivers pulses tunable over the wavelength range 450-2000 nm. These pulses are synchronized to the storage-ring RF clock with long-term stability better than 10 ps RMS. Monochromatic experimental capability with Biosafety Level 3 certification has been retained.