Signal Transduction in an Enzymatic Photoreceptor Revealed by Cryo-Electron Microscopy
Location
Poster #6
Start Date
26-4-2024 12:00 PM
Department
Chemistry
Abstract
Cryogenic electron microscopy (cryo-EM) enables the visualization of protein structures at near-atomic resolution, providing valuable insights into the overall architecture and conformational changes of macromolecules. Phytochromes are red light photoreceptors found in plants, fungi, and bacteria. They regulate a variety of important physiological responses such as seed germination and shade avoidance in plants. Their role in non-photosynthetic bacteria is unclear. Phytochromes are composed of a photosensory core module (PCM) covalently attached to the C-terminal effector domain, usually histidine kinase (HK). Molecular structures of the full-length protein, containing the HK domain have not been determined with X-ray crystallography as the intact protein is difficult to crystallize. The aim of this experiment was to obtain cryo-EM structures of the full-length wild-type bacteriophytochrome from non-photosynthetic myxobacterium Stigmatella aurantiaca in both its Pr and Pfr states. This protein was previously isolated in our laboratory and successfully studied with X-ray crystallography. However, only truncated variants of this protein, lacking the C-terminal effector domain, would crystallize, limiting the studies of the intact protein. The protein samples were purified, concentrated and immediately frozen at -80°C to be shipped to the Simons Electron Microscopy Center (SEMCO) of the New York Structural Biology Center (NYSBC). The protein samples were then vitrified by submerging the samples in liquid ethane. The vitrified samples were then subjected to an electron beam and the resulting micrographs were then used to reconstruct the molecule. CryoSPARC (v4.2.1) was used to reconstruct the molecule. Our study generated cryo-EM structures of the full-length wild-type SaBphP in both its Pr and Pfr state at 4.13Å and 3.75Å, respectively. A distinct heterodimer was also identified and imaged at 3.75Å and postulated to be a signal transduction intermediate. The active state of the SaBphP2 is still unknown but can be identified with a Phos-tag assay. The combination of cryo-EM and a Phos-tag assay represents a powerful approach for studying protein phosphorylation and its impact on protein structure and function. The Phos-tag technology utilizes a phosphate-binding tag that selectively binds to phosphorylated residues on proteins. By employing this assay in conjunction with cryo-EM, it becomes possible to identify and localize phosphorylation sites within protein structures. Our research will utilize this approach in the future to understand the phosphorylation-dependent protein dynamics of the signaling pathway that includes the SaBphP2.
Faculty Sponsor
Emina Stojkovic
Signal Transduction in an Enzymatic Photoreceptor Revealed by Cryo-Electron Microscopy
Poster #6
Cryogenic electron microscopy (cryo-EM) enables the visualization of protein structures at near-atomic resolution, providing valuable insights into the overall architecture and conformational changes of macromolecules. Phytochromes are red light photoreceptors found in plants, fungi, and bacteria. They regulate a variety of important physiological responses such as seed germination and shade avoidance in plants. Their role in non-photosynthetic bacteria is unclear. Phytochromes are composed of a photosensory core module (PCM) covalently attached to the C-terminal effector domain, usually histidine kinase (HK). Molecular structures of the full-length protein, containing the HK domain have not been determined with X-ray crystallography as the intact protein is difficult to crystallize. The aim of this experiment was to obtain cryo-EM structures of the full-length wild-type bacteriophytochrome from non-photosynthetic myxobacterium Stigmatella aurantiaca in both its Pr and Pfr states. This protein was previously isolated in our laboratory and successfully studied with X-ray crystallography. However, only truncated variants of this protein, lacking the C-terminal effector domain, would crystallize, limiting the studies of the intact protein. The protein samples were purified, concentrated and immediately frozen at -80°C to be shipped to the Simons Electron Microscopy Center (SEMCO) of the New York Structural Biology Center (NYSBC). The protein samples were then vitrified by submerging the samples in liquid ethane. The vitrified samples were then subjected to an electron beam and the resulting micrographs were then used to reconstruct the molecule. CryoSPARC (v4.2.1) was used to reconstruct the molecule. Our study generated cryo-EM structures of the full-length wild-type SaBphP in both its Pr and Pfr state at 4.13Å and 3.75Å, respectively. A distinct heterodimer was also identified and imaged at 3.75Å and postulated to be a signal transduction intermediate. The active state of the SaBphP2 is still unknown but can be identified with a Phos-tag assay. The combination of cryo-EM and a Phos-tag assay represents a powerful approach for studying protein phosphorylation and its impact on protein structure and function. The Phos-tag technology utilizes a phosphate-binding tag that selectively binds to phosphorylated residues on proteins. By employing this assay in conjunction with cryo-EM, it becomes possible to identify and localize phosphorylation sites within protein structures. Our research will utilize this approach in the future to understand the phosphorylation-dependent protein dynamics of the signaling pathway that includes the SaBphP2.