Ribbon diagram of ensemble of five ROSETTA models of full length RbfA with the structured core in black and the unstructured C-terminus in red. Figure provided by Masayori Inouye.
Protein structure determination by NMR spectroscopy places stringent demands on sample production. Sample preparation must be optimized to produce large amounts of stable protein over extended periods of data acquisition. Solubility enhancement tags (SETs) used during expression and purification are typically removed to avoid excess NMR signals. Since many proteins precipitate after tag removal, researchers have resorted to either using the smallest possible tags or exploring segmental labeling methods to introduce non-isotopically labeled tags which are not observed in the NMR data.
Inouye and colleagues (PSI NESG) present a new approach that uses protein splicing to introduce an invisible tag. Using tandem repeats of protein S (PrS2), a SET from Myxococcus xanthus, the authors developed a dual plasmid system for sample production. The first plasmid is used to express PrS2 fused to the N-terminal region of an intein in non-labeled medium. In parallel, the second plasmid is used to express PrS2 fused to both the C-terminus of the intein and a target protein in labeled media. Next, the soluble fractions of both cultures are mixed and intein ligation is initiated which, after protein splicing, results in the fusion of the unlabeled PrS2-tag to the labeled target protein.
The authors tested their Protein S Tag-Intein (PSTI) approach on ribosome binding factor A (RbfA) from Escherichia coli, a protein which previously required a C-terminal truncation (RbfAΔ25) to produce a stable sample for NMR analysis. After confirming that the PrS2-tag does not induce any conformational changes in RbfAΔ25, the authors produced full-length RbfA with the invisible SET. While they observed a dramatic increase in solubility for RbfA tagged with the invisible PrS2-tag, the NMR data were not of sufficient enough quality to assign the C-terminus. The authors found that a single PrS tag in place of the tandem PrS2-tag improved the data quality by reducing the overall molecular weight. They were finally able to assign the C-terminus of RbfA, which was found to be dynamically unstructured, presumably due to the absence of binding partners. Overall, the PSTI method has several distinct advantages and promises to expand the range of proteins accessible by NMR.