Bacteriophytochromes (BphPs) are photoreceptors that regulate a wide range of biological mechanisms via red light–absorbing (Pr)–to–far-red light–absorbing (Pfr) reversible photoconversion. The structural dynamics underlying Pfr-to-Pr photoconversion in a liquid solution phase are not well understood. We used time-resolved x-ray solution scattering (TRXSS) to capture light-induced structural transitions in the bathy BphP photosensory module of
Pseudomonas aeruginosa
. Kinetic analysis of the TRXSS data identifies three distinct structural species, which are attributed to lumi-F, meta-F, and Pr, connected by time constants of 95 μs and 21 ms. Structural analysis based on molecular dynamics simulations shows that the light activation of PaBphP accompanies quaternary structural rearrangements from an “II”-framed close form of the Pfr state to an “O”-framed open form of the Pr state in terms of the helical backbones. This study provides mechanistic insights into how modular signaling proteins such as BphPs transmit structural signals over long distances and regulate their downstream biological responses.
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.
Here, we introduce the nanoparticle-aided
cryo-electron microscopy
sampling (NACS) method to access the conformational distribution of
a protein molecule. Two nanogold particles are labeled at two target
sites, and the interparticle distance is measured as a structural
parameter via cryo-electron microscopy (cryo-EM). The key aspect of
NACS is that the projected distance information instead of the global
conformational information is extracted from each protein molecule.
This is possible because the contrast provided by the nanogold particles
is strong enough to provide the projected distance, while the protein
itself is invisible due to its low contrast. We successfully demonstrate
that various protein conformations, even for small or disordered proteins,
which generally cannot be accessed via cryo-EM, can be captured. The
demonstrated method with the potential to directly observe the conformational
distribution of such systems may open up new possibilities in studying
their dynamics at a single-molecule level.
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