Site-specific labeling of SBDS to monitor interactions with the 60S ribosomal subunit.

ABSTRACT

The Shwachman-Diamond syndrome (SDS) is a rare inherited ribosomopathy that is predominantly caused by mutations in the Shwachman-Bodian-Diamond Syndrome gene (SBDS). SBDS is a ribosomal maturation factor that is essential for the release of eukaryotic translation initiation factor 6 (eIF6) from 60S ribosomal subunits during the late stages of 60S maturation. Release of eIF6 is critical to permit inter-subunit interactions between the 60S and 40S subunits and to form translationally competent 80S monosomes. SBDS has three key domains that are highly flexible and adopt varied conformations in solution. To better understand the domain dynamics of SBDS upon binding to 60S and to assess the effects of SDS-disease specific mutations, we aimed to site-specifically label individual domains of SBDS. Here we detail the generation of a fluorescently labeled SBDS to monitor the dynamics of select domains upon binding to 60S. We describe the incorporation of 4-azido-l-phenylalanine (4AZP), a noncanonical amino acid in human SBDS. Site-specific labeling of SBDS using fluorophore and assessment of 60S binding activity are also described. Such labeling approaches to capture the interactions of individual domains of SBDS with 60S are also applicable to study the dynamics of other multi-domain proteins that interact with the ribosomal subunits.