Beyond Phosphate Esters: Synthesis of Unusually Phosphorylated Peptides and Proteins for Proteomic Research

PermaPhosSer: autonomous synthesis of functional, permanently phosphorylated proteins

“…Methylene phosphonates like nhpSer have reported pKa2 values of ∼7-8, compared for ∼5-6 for phosphates, 5 leading to concerns about the ability of nhpSer to mimic pSer. However, we found no studies that directly measured the pKa2 value of nhpSer in a protein.…”

“…2). Next, a combinatorial FrbABCDE library was created containing all permutations of the five Frb genes under the control of the five promoter variants (5 5 = 3025 pathway assemblies, Fig. 2B).…”

“…1,2 In humans, over 75% of proteins are reversibly phosphorylated at one or more sites, with nearly 80% of these being phosphoserine (pSer) modifications, 3 on which we focus here. Two factors have greatly limited efforts to define the interactions and functions of specific phospho-proteins: (1) the challenge of making specific homogeneous, and quantitatively phosphorylated protein forms for in vitro study and (2) the challenge of the transient nature of phosphorylation in the cellular milieu due to phosphatase activity 4,5 . The first challenge has now been well-addressed through genetic code expansion (GCE) systems that enable efficient, translational incorporation of pSer at genetically programmed codons into recombinant proteins produced in E. coli.…”

“…stable) analogs or mimics of phosphorylated amino acids (Fig. 1A) have been indispensable for in vitro and in cell studies, for creating inhibitors of phosphoprotein-binders, kinases and phosphatases that can serve as biochemical tools and potential therapeutics 4,5,[9][10][11] , and for making stable antigens for raising antibodies to recognize specific phosphoproteins 12 . The practical utility of such phosphomimetics hinges both on how well they faithfully recapitulate the effects of phosphorylation, and on the ease with which they can be quantitatively incorporated into proteins of interest.…”

“…Because the electronegative F-atoms of F2-nhpSer (i) can be H-bond acceptors like the pSer -oxygen and (ii) lower the phosphonate pKa2 from in the 7 to 8 range to near 5.5 so that its charge at physiological pH is ~ -2 (like pSer), it is generally considered a better analog than nhpSer. In fact, reviews give the impression that nhpSer is not a useful mimic for these reasons 5,23 , yet the effectiveness of both has been established in the few studies in which they were incorporated into synthetic peptides 12,[24][25][26][27] and into proteins via semi-synthetic methods such as expressed protein ligation (EPL) [28][29][30][31] . For example, proteins Histone 2A 30 and AANAT 28 with nhpSer (but not Asp/Glu) installed at their N-termini by EPL were able to pull down 14-3-3, a pSer/pThr specific binding protein, from cellular extracts.…”