The study of multidomain or large proteins in solution by NMR spectroscopy has been made possible in recent years by the development of new spectroscopic methods. However, resonance overlap found in large proteins remains a limiting factor, making resonance assignments and structure determination of large proteins very difficult. In this study, we present an expressed protein ligation protocol that can be used for the segmental isotopic labeling of virtually any multidomain or high molecular mass protein, independent of both the folding state and the solubility of the protein fragments, as well as independent of whether the fragments are interacting. The protocol was applied successfully to two different multidomain proteins containing RNA recognition motifs (RRMs), heterogeneous nuclear ribonucleoprotein L and Npl3p. High yields of segmentally labeled proteins could be obtained, allowing characterization of the interdomain interactions with NMR spectroscopy. We found that the RRMs of heterogeneous nuclear ribonucleoprotein L interact, whereas those of Npl3p are independent. Subsequently, the structures of the two RRMs of Npl3p were determined on the basis of samples in which each RRM was expressed individually. The two Npl3p RRMs adopt the expected beta alpha beta beta alpha beta fold.
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Evidence ID | Analyze ID | Gene/Complex | Systematic Name/Complex Accession | Qualifier | Gene Ontology Term ID | Gene Ontology Term | Aspect | Annotation Extension | Evidence | Method | Source | Assigned On | Reference |
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Evidence ID | Analyze ID | Gene | Gene Systematic Name | Phenotype | Experiment Type | Experiment Type Category | Mutant Information | Strain Background | Chemical | Details | Reference |
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Evidence ID | Analyze ID | Gene | Gene Systematic Name | Disease Ontology Term | Disease Ontology Term ID | Qualifier | Evidence | Method | Source | Assigned On | Reference |
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Evidence ID | Analyze ID | Regulator | Regulator Systematic Name | Target | Target Systematic Name | Direction | Regulation of | Happens During | Regulator Type | Direction | Regulation Of | Happens During | Method | Evidence | Strain Background | Reference |
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Site | Modification | Modifier | Source | Reference |
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Evidence ID | Analyze ID | Interactor | Interactor Systematic Name | Interactor | Interactor Systematic Name | Allele | Assay | Annotation | Action | Phenotype | SGA score | P-value | Source | Reference | Note |
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Evidence ID | Analyze ID | Interactor | Interactor Systematic Name | Interactor | Interactor Systematic Name | Assay | Annotation | Action | Modification | Source | Reference | Note |
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Complement ID | Locus ID | Gene | Species | Gene ID | Strain background | Direction | Details | Source | Reference |
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Evidence ID | Analyze ID | Dataset | Description | Keywords | Number of Conditions | Reference |
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