Structure and function of Colicin A / Cai and PutP studied by site directed spin labeling EPR spectroscopy

In this work 3 different proteins are subjected to investigations on their structural, dynamic and functional properties by SDSL EPR spectroscopy, combined with in silico structure prediction and modeling: the pore-forming bacterial toxin colicin A in its membrane-bound form and its corresponding immunity protein Cai, and the Na+/proline symporter PutP. Colicin A (ColA) is a plasmid-encoded water-soluble pore-forming toxin produced by certain E. coli strains that kills unprotected cells of related strains by inserting a pore-forming subdomain into the cytoplasmic membrane to form voltage-dependent ion channels. Detailed structural data for the membrane-bound channel, in the closed as well as in the open state, is still missing, thus in the present study, the in vitro investigation by site-directed spin labeling and EPR spectroscopy has been substantially extended. The results indicate that a larger fraction of the protein than previously suggested penetrates into the hydrophobic core of the membrane, and distance measurements by pulse and cw EPR spectroscopy provide evidence that ColA in lipid bilayer membranes forms an oligomeric structure. Pulse EPR distance measurements under in vivo conditions reveal clear indications for an oligomeric ColA structure also in vivo. The results of all EPR measurements were combined to construct a dimer model for the colicin A closed channel state conformation. The immunity protein Cai, an integral inner membrane protein, protects the producing E. coli cell from the cytotoxic activity of its corresponding toxin (colicin A), by preventing channel opening by a yet unknown mechanism. ESR measurements for single spin label probes attached to ColA in the presence and absence of the immunity protein Cai reveal a clear influence on the ColA helices of the pore-forming domain in the presence of Cai as previously postulated. The data suggest that Cai induces a conformational change in/for the voltage sensor helix H6 of ColA, forming a “locked” inactive channel conformation that is not capable of voltage sensing and channel opening. Initial experiments with spin labeled wt-Cai in the presence and absence of unlabeled ColA suggest a more compact structure in the presence of ColA. PutP is an integral membrane protein located in the cytoplasmic membrane of E. coli, being responsible for the coupled transport of Na+ and proline in a 1:1 stoichiometry. It belongs to the family of sodium solute symporters (SSSF). Three dimensional structural data for PutP are at the moment not available, but a homology model has been developed based on the crystal structure of another member of this protein family, the Na+/galactose symporter vSGLT of Vibrio parahaemolyticus. The observed periodicity in spin label mobility and polarity measurements suggest a secondary structure of the extracellular Loop eL4 of PutP of two α-helical segments eL4a and eL4b, and imply the idea of eL4 functioning as an external gate to the SSSF. The ligand-induced changes observed in mobility, polarity and accessibility upon substrate binding support this notion, thus providing further insights into the mechanistic basis of sodium solute symport

[¹⁸F]<i>p</i>FBC, a Covalent CLIP-Tag Radiotracer for Detection of Viral Reporter Gene Transfer in the Murine Brain

Preclinical models of neurological diseases and gene therapy are essential for neurobiological research. However, the evaluation of such models lacks reliable reporter systems for use with noninvasive imaging methods. Here, we report the development of a reporter system based on the CLIP-tag enzyme and [18F]pFBC, an 18F-labeled covalent CLIP-tag-ligand synthesized via a DoE-optimized and fully automated process. We demonstrated its specificity using a subcutaneous xenograft model and a model of viral vector-mediated brain gene transfer by engineering HEK293 cells and striatal neurons to express membrane-tethered CLIP-tag protein. After in vitro characterization of the reporter, mice carrying either CLIP-tag expressing or control subcutaneous xenografts underwent dynamic [18F]pFBC PET imaging. The CLIP-tag expressing xenografts showed a significantly higher uptake than control xenografts (tumor-to-muscle ratio 5.0 vs 1.7, p = 0.0379). In vivo, metabolite analysis by radio-HPLC from plasma and brain homogenates showed only one radio-metabolite in plasma and none in the brain. In addition, [18F]pFBC showed fast uptake and rapid clearance from the brain in animals injected with adeno-associated virus (AAV)-CLIP in the right striatum but no right-to-left (R-L) uptake difference in the striata in the acquired PET data. In contrast, autoradiography showed a clear accumulation of radioactivity in the AAV-CLIP-injected right striatum compared to the sham-injected left striatum control. CLIP-tag expression and brain integrity were verified by immunofluorescence and light sheet microscopy. In conclusion, we established a novel reporter gene system for PET imaging of gene expression in the brain and periphery and demonstrated its potential for a wide range of applications, particularly for neurobiological research and gene therapy with viral vectors