RESUMEN
Genetic code expansion allows modification of the physical and chemical properties of proteins by the site-directed insertion of noncanonical amino acids. Here we exploit this technology for measuring nanometer-scale distances in proteins. (2,2'-Bipyridin-5-yl)alanine was incorporated into the green fluorescent protein (GFP) and used as an anchoring point for Cu(II) to create a spin-label. The incorporation of (2,2'-bipyridin-5-yl)alanine directly into the protein resulted in a high-affinity binding site for Cu(II) capable of outcompeting other binding positions in the protein. The resulting Cu(II)-spin label is very compact and not larger than a conventional amino acid. By using 94 GHz electron paramagnetic resonance (EPR) pulse dipolar spectroscopy we have been able to determine accurately the distance between two such spin-labels. Our measurements revealed that GFP dimers can adopt different quaternary conformations. The combination of spin-labeling using a paramagnetic nonconventional amino acid with high-frequency EPR techniques resulted in a sensitive method for studying the structures of proteins.
