Optimization and CH methionine labeling of a signaling competent neurotensin receptor 1 variant for NMR studies.

Neurotensin is a 13-residue peptide that acts as a neuromodulator of classical neurotransmitters such as dopamine and glutamate in the mammalian central nervous system, mainly by activating the G protein-coupled receptor (GPCR), neurotensin receptor 1 (NTS). Agonist binding to GPCRs shifts the conformational equilibrium of the transmembrane helices towards distinct, thermodynamically favorable conformations that favor effector protein interactions and promotes cell signaling. The introduction of site specific labels for NMR spectroscopy has proven useful for investigating this dynamic process, but the low expression levels and poor stability of GPCRs is a hindrance to solution NMR experiments.

A Novel Ultra-Stable, Monomeric Green Fluorescent Protein For Direct Volumetric Imaging of Whole Organs Using CLARITY.

Recent advances in thick tissue clearing are enabling high resolution, volumetric fluorescence imaging of complex cellular networks. Fluorescent proteins (FPs) such as GFP, however, can be inactivated by the denaturing chemicals used to remove lipids in some tissue clearing methods. Here, we solved the crystal structure of a recently engineered ultra-stable GFP (usGFP) and propose that the two stabilising mutations, Q69L and N164Y, act to improve hydrophobic packing in the core of the protein and facilitate hydrogen bonding networks at the surface, respectively.

Targeting Lyn tyrosine kinase through protein fusions encompassing motifs of Cbp (Csk-binding protein) and the SOCS box of SOCS1.

The tyrosine kinase Lyn is involved in oncogenic signalling in several leukaemias and solid tumours, and we have previously identified a pathway centred on Cbp [Csk (C-terminal Src kinase)-binding protein] that mediates both enzymatic inactivation, as well as proteasomal degradation of Lyn via phosphorylation-dependent recruitment of Csk (responsible for phosphorylating the inhibitory C-terminal tyrosine of Lyn) and SOCS1 (suppressor of cytokine signalling 1; an E3 ubiquitin ligase). In the present study we show that fusing specific functional motifs of Cbp and domains of SOCS1 together generates a novel molecule capable of directing the proteasomal degradation of Lyn. We have characterized the binding of pY (phospho-tyrosine) motifs of Cbp to SFK (Src-family kinase) SH2 (Src homology 2) domains, identifying those with high affinity and specificity for the SH2 domain of Lyn and that are preferred substrates of active Lyn.

Purification, crystallization, small-angle X-ray scattering and preliminary X-ray diffraction analysis of the SH2 domain of the Csk-homologous kinase.

The C-terminal Src kinase (Csk) and Csk-homologous kinase (CHK) are endogenous inhibitors of the proto-oncogenic Src family of protein tyrosine kinases (SFKs). Phosphotyrosyl peptide binding to their Src-homology 2 (SH2) domains activates Csk and CHK, enhancing their ability to suppress SFK signalling; however, the detailed mechanistic basis of this activation event is unclear. The CHK SH2 was expressed in Escherichia coli and the purified protein was characterized as monomeric by synchrotron small-angle X-ray scattering in-line with size-exclusion chromatography.

Bacterial expression and purification of active hematopoietic cell kinase.

Src family kinases (SFKs) are traditionally purified from eukaryotic expression systems. These expression systems can be costly, yield heterogeneously phosphorylated protein samples and present difficulties when metabolic labeling is required for structural studies. Therefore, many attempts have been made to develop bacterial purification systems for SFKs.