Background: Cyclic di-AMP inactivates the potassium transport activity of KtrA. Results: Cyclic di-AMP binding to KrtA induced conformational changes. Conclusion: Cyclic di-AMP selectively binds to the KtrA RCK_C domain and signals the inactivation of potassium transport. Significance: The molecular basis for the role of cyclic di-AMP in potassium channel activity was investigated.

Ultrafast motion of molecules, particularly the coherent motion, has been intensively investigated as a key factor guiding the reaction pathways. Recently, X-ray free-electron lasers (XFELs) have been utilized to elucidate the ultrafast motion of molecules. However, the studies on proteins using XFELs have been typically limited to the crystalline phase, and proteins in solution have rarely been investigated. Here we applied femtosecond time-resolved X-ray solution scattering (fs-TRXSS) and a structure refinement method to visualize the ultrafast motion of a protein. We succeeded in revealing detailed ultrafast structural changes of homodimeric hemoglobin involving the coherent motion. In addition to the motion of the protein itself, the time-dependent change of electron density of the hydration shell was tracked. Besides, the analysis on the fs-TRXSS data of myoglobin allows for observing the effect of the oligomeric state on the ultrafast coherent motion.

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Salt bridge, one of the representative structural factors established by non-covalent interactions, plays a crucial role in stabilizing the structure and regulating the protein function, but its role in dynamic...

Photoactive yellow protein (PYP) induces negative phototaxis in Halorhodospira halophila via photoactivation triggered by light-mediated chromophore isomerization. Chromophore isomerization proceeds via a volume-conserving isomerization mechanism due to the hydrogen-bond network and steric constraints inside the protein, and causes significant conformational changes accompanied by N-terminal protrusion. However, it is unclear how the structural change of the chromophore affects the remote N-terminal domain. To understand photocycle-related structural changes, we investigated the structural aspect of chromophore removal in PYP because it possesses a disrupted hydrogen-bond network similar to that in photocycle intermediates. A comparison of the structural aspects with those observed in the photocycle would give a clue related to the structural change mechanism in the photocycle Chromophore removal effects were assessed via UV-vis spectroscopy, circular dichroism, and X-ray solution scattering. Molecular shape reconstruction and an experiment-restrained rigid-body molecular dynamics simulation based on the scattering data were performed to determine protein shape, size, and conformational changes upon PYP bleaching. Data show that chromophore removal disrupted the holo-PYP structure, resulting in a small N-terminal protrusion, but the extent of conformational changes was markedly less than those in the photocycle. This indicates that disruption of the hydrogen-bond network alone in bleached PYP does not induce the large conformational change observed in the photocycle, which thus must result from the organized structural transition around the chromophore triggered by chromophore photoisomerization along with disruption of the hydrogen-bond network between the chromophore and the PYP core.

A fundamental problem in biological sciences is understanding how macromolecular machines work and how the structural changes of a molecule are connected to its function. Time-resolved techniques are vital in this regard and essential for understanding the structural dynamics of biomolecules. Time-resolved small- and wide-angle X-ray solution scattering has the capability to provide a multitude of information about the kinetics and global structural changes of molecules under their physiological conditions. However, standard protocols for such time-resolved measurements often require significant amounts of sample, which frequently render time-resolved measurements impossible. A cytometry-type sheath co-flow cell, developed at the BioCARS 14-ID beamline at the Advanced Photon Source, USA, allows time-resolved pump–probe X-ray solution scattering measurements to be conducted with sample consumption reduced by more than ten times compared with standard sample cells and protocols. The comparative capabilities of the standard and co-flow experimental setups were demonstrated by studying time-resolved signals in photoactive yellow protein.

Using various spectroscopic techniques such as UV-visible spectroscopy, circular dichroism spectroscopy, NMR spectroscopy, small-angle X-ray scattering, transient grating, and transient absorption techniques, we investigated how cell-mimetic environments made by crowding influence the photocycle of photoactive yellow protein (PYP) in terms of the molecular volume change and kinetics. Upon addition of molecular crowding agents, the ratio of the diffusion coefficient of the blue-shifted intermediate (pB) to that of the ground species (pG) significantly changes from 0.92 and approaches 1.0. This result indicates that the molecular volume change accompanied by the photocycle of PYP in molecularly crowded environments is much smaller than that which occurs in vitro and that the pB intermediate under crowded environments favors a compact conformation due to the excluded volume effect. The kinetics of the photocycle of PYP in cell-mimetic environments is greatly decelerated by the dehydration, owing to the interaction between the protein and small crowding agents, but is barely affected by the excluded volume effect. The results lead to the inference that the signaling transducer of PYP may not necessarily utilize the conformational change of PYP to sense the signaling state.

Obtaining the heterogeneous conformation of small proteins is important for understanding their biological role, but it is still challenging. Here, we developed a multi-tilt nanoparticle-aided cryo-electron microscopy sampling (MT-NACS) technique that enables the observation of heterogeneous conformations of small proteins and applied it to calmodulin. By imaging the proteins labeled by two gold nanoparticles at multiple tilt angles and analyzing the projected positions of the nanoparticles, the distributions of 3D interparticle distances were obtained. From the measured distance distributions, the conformational changes associated with Ca 2+ binding and salt concentration were determined. MT-NACS was also used to track the structural change accompanied by the interaction between amyloid-beta and calmodulin, which has never been observed experimentally. This work offers an alternative platform for studying the functional flexibility of small proteins.