Synthesis and Evaluation of Non‐Hydrolyzable Phospho‐Lysine Peptide Mimics

Abstract

Invited for the cover of this issue is the group of Christian P. R. Hackenberger at the Leibniz‐Forschungsinstitut für Molekulare Pharmakologie and the Humboldt‐Universität zu Berlin. The image depicts a phospho‐lysine peptide mimic which reflects a phosphorylated lysine but is not identical. Read the full text of the article at 10.1002/chem.202003947.

PermaPhosSer: autonomous synthesis of functional, permanently phosphorylated proteins

“…6). This pKa2 of nhpSer is similar to the value of 7.08 seen for a phosphonate analog of phospho-lysine 41 . These data imply that at pH 7.4, nhpSer in this context exhibits an average charge of -1.7 (~71% dianonic), compared to -2.0 for pSer, whereas the carboxylate of Asp/Glu is -1.0.…”

“…To directly measure their pKa2, we used the above-described SUMO-HSPB6 fusion protein with pSer and nhpSer at Ser16 (Fig. 4A) and followed the 31 P NMR chemical shifts as a function of pH 41,42 . We observed a well-defined 31 P peak for nhpSer and pSer containing proteins, and consistent with previous work, increasing pH resulted in an upfield 31 P chemical shift for nhpSer and a downfield shift for pSer 41 , with the inflection points indicating a pKa2 of 7.00 ± 0.05 for nhpSer and 5.78 ± 0.06 for pSer (Fig.…”

Autonomous Synthesis of Functional, Permanently Phosphorylated Proteins for Defining the Interactome of Monomeric 14-3-3ζ

“…Whether this p K a2 is closer to 7 or to 8 changes its protonation state at physiologic state quite notably, yet we found no studies that have measured this directly in the context of a protein. To do this, we used the above-described SUMO-HSPB6 fusion protein with pSer and nhpSer at Ser16 (Figure A) and followed the 31 P NMR chemical shifts as a function of pH. , This construct was chosen because it was highly soluble, stable, and small (i.e., suitable for NMR) and also because the phospho-site is within an unstructured segment such that phosphorus resonance changes should reflect the protonation state rather than changes in the protein conformation. We observed a well-defined 31 P peak for nhpSer- and pSer-containing proteins, and consistent with previous work, increasing pH resulted in an upfield 31 P chemical shift for nhpSer and a downfield shift for pSer, with the inflection points indicating a p K a2 of 7.00 ± 0.05 for nhpSer and 5.78 ± 0.06 for pSer (Figures B and S7).…”

“…47,48 This construct was chosen because it was highly soluble, stable, and small (i.e., suitable for NMR) and also because the phospho-site is within an unstructured segment such that phosphorus resonance changes should reflect the protonation state rather than changes in the protein conformation. We observed a well-defined 31 P peak for nhpSer-and pSer-containing proteins, and consistent with previous work, increasing pH resulted in an upfield 31 P chemical shift for nhpSer and a downfield shift for pSer, 47 with the inflection points indicating a pK a2 of 7.00 ± 0.05 for nhpSer and 5.78 ± 0.06 for pSer (Figures 4B and S7). These data imply that at pH 7.4, nhpSer in this context exhibits an average charge of −1.7 (∼71% dianonic), compared to −2.0 for pSer, whereas that for the carboxylate of Asp/Glu is −1.0.…”

Genetic Encoding of Phosphorylated Amino Acids into Proteins

“…human proteins identified. 19 Just as for pArg (19), the phosphorylation of lysine changes its net charge from +1 to −1 at physiologic pH because the N ε remains protonated at physiologic pH (pK a ∼ 10), 20,305 and such charge reversals can readily impact protein structure and function. pLys (17) is rapidly hydrolyzed at acidic pH like other amino acids with phosphoramidate (N−P) bonds (t 1/2 < 1 min for N-(n-butyl) phosphoramidate) and is moderately stable at physiologic pH.…”