As Jung-Min Kee and Tom W. Muir noted in their recent review article (ACS Chem. Biol., 2012, 7 (1), pp 44–51), 2012 marked the 50th anniversary of the discovery of histidine phosphorylation in proteins. Post-translational phosphorylation of His residues at either the N-1 or N-3 positions of the imidazole group has been well recognized as an important signaling mechanism in prokaryotes and lower eukaryotes, and now is being associated with mammalian cellular processes, cancer, and inflammation. However its chemical lability especially at low pH has hindered its detection and study.
The RcsD protein of E. coli is part of a phosphorelay signal transduction system that accepts phosphate from RcsC on the His residue in the following target sequence: SDFAALAQTAHRLKGVFAMLN. Assume you have synthesized this sequence and achieved in vitro phosphorylation of its His by transfer from RcsC. Chemical characterization of both forms of the oligopeptide reveals the following pKas:
N- and C-terminal, 9.5 and 4.5, respectively; Asp side chain 3.5; Lys side chain 10.5; Arg side chain 12.0; and His side chain 6.0. Upon phosphorylation, the His imidazole pKa shifts to 6.5; the phospho group has pKas of 2.0 and 6.8.
a. On the single letter sequences of the His- and pHis-oligopeptides, indicate the charges the residues will carry at the pI points for the oligopeptides. Indicate partial charges (i.e., 10% - 90% charge) with ?+ and ?-.
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Calculate the pI of each oligopeptide.
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If the two oligopeptides were chromatographed on a DEAE-matrix at pH 7.5, in what order would they emerge? Explain why.
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If the oligopeptides were reacted with cyanogen bromide, would their separation on the DEAE be changed? Explain why.