ABSTRACT
Evidence of a complex formation is a crucial step in the structural studies of ligand-receptor interactions. Here we presented a simple and fast approach for qualitative screening of the complex formation between the chimeric extracellular domain of the nicotinic acetylcholine receptor (α7-ECD) and three-finger proteins. Complex formation of snake toxins α-Bgtx and WTX, as well as of recombinant analogs of human proteins Lynx1 and SLURP-1, with α7-ECD was confirmed using fluorescently labeled ligands and size-exclusion chromatography with simultaneous absorbance and fluorescence detection. WTX/α7-ECD complex formation also was confirmed by cryo-EM. The proposed approach could easily be adopted to study the interaction of other receptors with their ligands.
Subject(s)
alpha7 Nicotinic Acetylcholine Receptor/chemistry , alpha7 Nicotinic Acetylcholine Receptor/metabolism , Animals , Bungarotoxins/chemistry , Bungarotoxins/metabolism , Carrier Proteins/chemistry , Carrier Proteins/metabolism , Carrier Proteins/ultrastructure , Chromatography, Gel , Cryoelectron Microscopy , Elapid Venoms/chemistry , Elapid Venoms/metabolism , Fluorescent Dyes , Humans , Ligands , Protein Interaction Domains and Motifs , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/metabolism , Recombinant Fusion Proteins/ultrastructure , Surface Plasmon Resonance , alpha7 Nicotinic Acetylcholine Receptor/ultrastructureABSTRACT
The structure of cytochrome c nitrite reductase from the bacterium Thioalkalivibrio nitratireducens was determined by cryo-electron microscopy (cryo-EM) at a 2.56 Å resolution. Possible structural heterogeneity of the enzyme was assessed. The backbone and side-chain orientations in the cryo-EM-based model are, in general, similar to those in the high-resolution X-ray diffraction structure of this enzyme.
