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Structural biology of the intrinsically disordered protein SH2B1


Project Description

Signalling between the outside of the cell and the inside uses a complicated network of interacting systems. One common signalling mechanism uses receptor tyrosine kinases: binding of a ligand to the receptor causes dimerization of the receptor, which leads to the intracellular kinase domain on one receptor phosphorylating the kinase domain on its neighbour and activating it. The intracellular phosphorylation is recognised by a specific binding domain, often an SH2 domain, which acts to relay the signalling downstream and ultimately to lead to metabolic changes in the cell. In many cases, there are extra proteins involved that modify the signal response. SH2B1 is one of these. It is a large protein (670 amino acids) that contains an SH2 domain and binds to phosphorylated receptors. It also contains a PH domain (binds to membranes) and a self-dimerisation domain. The rest of the protein appears to be intrinsically disordered. Intrinsically disordered proteins (IDPs) often have weak intramolecular binding between folded domains and disordered sections, which alter the availability of binding sites, bring different parts of the protein closer together, create new interaction sites, and regulate protein-protein association (Williamson & Potts Biochem Soc Trans, 2012, 40:945-949). It is therefore likely that the IDP regions of SH2B1 have functions in controlling the association of ligands to target receptors, dependent on intramolecular interactions between IDP regions and folded domains. For example, there has been a publication describing a likely interaction between the SH2 domain and a sequence within residues 1-247.
SH2B1 binds to the insulin receptor, and also to several other related receptors including the leptin receptor, which has a function in regulating eating and obesity. Deletion of the gene for SH2B1 leads to severe obesity in mice, insulin resistance, age-dependent hyperinsulinaemia, hyperphagia [overeating], diabetes and glucose intolerance (DC Ren et al., Cell Metabol., 2005, 2:95-104; M Li et al., Endocrinology, 2006, 147:2163-2170.) It is therefore important, but its exact function is not well understood. The aim of the project is to study intramolecular interactions within SH2B1 and gain insights into how it works.

Outline of work to be undertaken

We will use polymerase chain reaction (PCR) to put restriction sites into the SH2B1 gene, cut out different parts of the gene, and insert them into E. coli, where they will be expressed with histidine tags so that we can use nickel affinity chromatography to purify the protein products. The proteins will be analysed by chromatography and NMR to see if they are folded. Folded regions may need some trimming at the ends to get them properly folded and without excessively long unfolded tails. They will then be expressed as isotopically labelled proteins and their NMR spectra will be assigned. The assignments will be used to define the secondary structure. Any novel proteins will have their structures determined.
We will analyse binding of IDP regions to the folded regions using chemical shift perturbation with 15N-labelled folded domains, which will give us interaction regions and affinities. Different lengths of IDP and different linker lengths will be studied to give us a clearer picture of intramolecular binding. If there is time, we will study dimerization of the dimerization domain and the effect of phosphorylation on affinities.


Science Graduate School

As a PhD student in one of the science departments at the University of Sheffield, you’ll be part of the Science Graduate School – a community of postgraduate researchers working across biology, chemistry, physics, mathematics and psychology. You’ll get access to training opportunities designed to support your career development by helping you gain professional skills that are essential in all areas of science. You’ll be able to learn how to recognise good research and research behaviour, improve your communication abilities and experience technologies that are used in academia, industry and many related careers. Visit http://www.sheffield.ac.uk/sgs to learn more.

Funding Notes

Entry requirements
First class or upper second 2(i) in a relevant subject. To formally apply for a PhD position, you must complete the University's application form using the following link: View Website

*All applicants should ensure that both references are uploaded onto their application as a decision will be unable to be made without this information*.


How good is research at University of Sheffield in Biological Sciences?

FTE Category A staff submitted: 44.90

Research output data provided by the Research Excellence Framework (REF)

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