Protein phosphorylation by semisynthesis: from paper to practice.
Deconvolution of specific phosphorylation events can be complicated by the reversibility of modification. Protein semisynthesis with phosphonate analogues offers an attractive approach to functional analysis of signaling pathways. In this technique, N- and C-terminal synthetic peptides containing nonhydrolyzable phosphonates at target residues can be ligated to recombinant proteins of interest. The resultant semisynthetic proteins contain site specific, stoichiometric phosphonate modifications and are completely resistant to phosphatases. Control of stoichiometry, specificity, and reversibility allows for complex signaling systems to be broken down into individual events and discretely examined. This chapter outlines the general methods and considerations for designing and carrying out phosphoprotein semisynthetic projects.
Szewczuk
Lawrence M
LM
Department of Pharmacology and Molecular Sciences, The Johns Hopkins University School of Medicine, Baltimore, Maryland, USA.
Tarrant
Mary Katherine
MK
Cole
Philip A
PA
D000409:Alanine / Q000031:analogs & derivatives / Q000737:chemistry; D000818:Animals; D006801:Humans; D047668:Inteins; D063065:Organophosphonates / Q000138:chemical synthesis* / Q000737:chemistry; D010649:Phenylalanine / Q000031:analogs & derivatives / Q000737:chemistry; D019606:Phosphoamino Acids / Q000737:chemistry*; D010748:Phosphopeptides / Q000138:chemical synthesis*; D010750:Phosphoproteins / Q000737:chemistry* / Q000378:metabolism*; D010766:Phosphorylation; D010768:Phosphoserine / Q000031:analogs & derivatives / Q000737:chemistry; D010769:Phosphothreonine / Q000031:analogs & derivatives / Q000737:chemistry; D011993:Recombinant Fusion Proteins / Q000737:chemistry* / Q000378:metabolism
D063065:Organophosphonates; D019606:Phosphoamino Acids; D010748:Phosphopeptides; D010750:Phosphoproteins; D011993:Recombinant Fusion Proteins; C499836:phosphonodifluoromethylene alanine; C089985:phosphonodifluoromethylphenylalanine; D010769:Phosphothreonine; D010768:Phosphoserine; D010649:Phenylalanine; D000409:Alanine