Question

5. Ser, Thr, and Tyr are well known targets for post-translational phosphorylation. A lesser known target is His [Kee and Muir, ACS Chem. Biol., 2012, 7 (1), pp 44–51], which can be phosphorylated at either the N-1 or N-3 positions of the imidazole group. One hindrance in studying this phosphorylation is its chemical lability, especially at low pH. Nevertheless, His phosphorylation is now recognized as an important signaling mechanism in prokaryotes and lower eukaryotes, and is being associated with mammalian cellular processes, cancer, and inflammation.

In E. coli the RcsD protein is part of a phosphorelay signal transduction system that accepts phosphate from RcsC, becoming phosphorylated on the His residue in the target sequence SDFAALAQTAHRLKGVFAMLN. Assume you have synthesized this sequence and achieved in vitro phosphorylation of the His by transfer from RcsC. Chemical characterization of the oligopeptide reveals the following pKas:

N- and C-terminal, 9.5 and 4.5, respectively; Asp side chain 4.3; Lys side chain 10.1; Arg side chain 12.0; and His side chain 5.5. Upon phosphorylation, the His imidazole pKa shifts to 6.5; the phospho group has pKas of 2.0 and 6.8.

a. Write the single letter sequences of the His- and pHis-oligopeptides and indicate above the sequence the charges the residues will carry at pH 7.0. Indicate partial charges (i.e., 10% - 90% charged) with δ+ and δ-.

b. Calculate the pI of each oligopeptide.

c. If a mixture of the His- and pHis-oligopeptides were passed through a column containing DEAE-matrix at pH 7.0, how would they behave? Explain why.

d. If a mixture of the two oligopeptides were reacted with cyanogen bromide followed by V8 protease digestion, could the His and pHis-containing peptides be separated by DEAE chromatography at pH 7.0? Explain why.

Answer 1

a) the sequence of peptide is

SDFAALAQTAHRLKGVFAMLN

Out of these the single letter notation for histidine amino acid = H

So we will put small "p" after H in the oligopeptide

SDFAALAQTAHpRLKGVFAMLN

b) For His -oligopeptide

the isoelectric point = (pKa of N-terminus + pKa of Lysine) / 2

Given : N- and C-terminal, 9.5 and 4.5, respectively, Lys side chain 10.1

So PI = 9.5 + 10.1 / 2 = 9.8

For phosph-Histidien peptide

The PI = (pKa of p-Histidine + pKa2 of pHis phosphorylated )

Given : His imidazole pKa shifts to 6.5; the phospho group has pKas of 2.0 and 6.8.

PI = 6.5 + 6.8 / 2 = 6.65

c) We know that DEAE is a positively charged resin and used to filter positive and negative peptides

So it lock the negative peptides

Out of His and phosphorlyated His oligo peptide

The His oligopeptide carries a positive charge

While due to phosphate group the p-His oligo peptide has a partial negative charge (at pH =7)

so DEAE will lock pHis oligo peptide and His oligo peptide will be passed out

d) The action of cyanogen bromide is on the carboxyl group of methionine followed by protease digestion.so it will cause cleavage of peptide it would not be possible to separate the two oliogpeptide