Synthetic probes of histidine phosphorylation: new reagents for systems biology and proteomics

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DNA sequencing has revealed that the human genome comprises only 30000 distinct proteins. That such a relatively small number of proteins can combine to form a human being is due to the vast number of ways in which they can be modified to control their function and behaviour. The dominant method by which this occurs is protein phosphorylation, a vast body of research in the last fifty years (now comprising several thousand publications/year) has demonstrated how modification of the amino acids serine, threonine and tyrosine control the physiology of the cell and whole organisms. Errors in these control and signalling pathways are implicated in many human diseases including cancer.There are twenty natural amino acids in total and many others can also be phosphorylated. We are interested in exploring the biochemistry of phosphorylated histidine which has been little studied to date. N-linked Phosphohistidine is less stable than its O-linked counterparts and it has not therefore been possible to acquire antibodies capable of identifying this modification. This, in turn, means that it has been impossible to assess how prevalent the modification is in cells and how it is involved in regulating complex biochemical processes. In this project we will generate the first class of such reagents thereby enabling a step change in research in this area.We will synthesize a range of stable analogues of phosphohistidine and incorporate these into short peptides. We will then evaluate the analogues by measuring how well they bind to a protein known to recognise and modify phosphohistidine-containing proteins. Following this we will screen libraries of peptide aptamers for aptamers which specifically recognise the analogue and thereby phosphohistidine. Such a tool will then be applied to the assay of other biological systems.