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Abstract

Site-specific labeling via disulfide bridges or thioether linkages are widely employed for paramagnetic probe attachment because cysteine residues can be selectively incorporated in protein. In order to investigate protein-protein and protein-ligand interactions, multiple paramagnetic data sets are required; however, finding suitable mutation sites is sometimes difficult, especially for small proteins. An alternative is to attach different paramagnetic probes to same site. Several CLaNP-5 derivatives have been synthesized for this purpose and one of them was tagged to a model protein. The HSQC spectra of methoxyl functionalized CLaNP-5 showed two sets of PCSs. This phenomenon could be explained by an interaction with a neighboring amino acid with the methoxyl group.