In-cell biochemistry using NMR spectroscopy.
Biochemistry and structural biology are undergoing a dramatic revolution. Until now, mostly in vitro techniques have been used to study subtle and complex biological processes under conditions usually remote from those existing in the cell. We developed a novel in-cell methodology to post-translationally modify interactor proteins and identify the amino ... acids that comprise the interaction surface of a target protein when bound to the post-translationally modified interactors. Modifying the interactor proteins causes structural changes that manifest themselves on the interacting surface of the target protein and these changes are monitored using in-cell NMR. We show how Ubiquitin interacts with phosphorylated and non-phosphorylated components of the receptor tyrosine kinase (RTK) endocytic sorting machinery: STAM2 (Signal-transducing adaptor molecule), Hrs (Hepatocyte growth factor regulated substrate) and the STAM2-Hrs heterodimer. Ubiquitin binding mediates the processivity of a large network of interactions required for proper functioning of the RTK sorting machinery. The results are consistent with a weakening of the network of interactions when the interactor proteins are phosphorylated. The methodology can be applied to any stable target molecule and may be extended to include other post-translational modifications such as ubiquitination or sumoylation, thus providing a long-awaited leap to high resolution in cell biochemistry.
Mesh Terms:
Adaptor Proteins, Signal Transducing, Biochemistry, Cytological Techniques, Dimerization, Endocytosis, Endosomal Sorting Complexes Required for Transport, Escherichia coli, Humans, Magnetic Resonance Spectroscopy, Models, Statistical, Phosphoproteins, Phosphorylation, Protein Processing, Post-Translational, Receptor Protein-Tyrosine Kinases, Ubiquitin
Adaptor Proteins, Signal Transducing, Biochemistry, Cytological Techniques, Dimerization, Endocytosis, Endosomal Sorting Complexes Required for Transport, Escherichia coli, Humans, Magnetic Resonance Spectroscopy, Models, Statistical, Phosphoproteins, Phosphorylation, Protein Processing, Post-Translational, Receptor Protein-Tyrosine Kinases, Ubiquitin
PLoS ONE
Date: Jul. 16, 2008
PubMed ID: 18626516
View in: Pubmed Google Scholar
Download Curated Data For This Publication
86072
Switch View:
- Interactions 2