Passage
Interactions between positively charged histones and negatively charged nucleic acids are altered by acetylation and methylation of histone subunits. These different post-translational modifications are recognized by histone-binding proteins such as BROMO domain Adjacent to Zinc finger 2B (BAZ2B) . BAZ2B has a net negative charge but contains a lysine-rich regulatory region between its two histone-binding domains.NMR data revealed potential interactions between BAZ2B and histone subunit H3 (Figure 1) . Binding was confirmed by isothermal titration calorimetry (ITC) , which measures enthalpy of binding when a protein solution is titrated with ligand injections of equal volume and concentration. Each injection results in a negative peak as heat is released. Figure 2 shows ITC results when wild-type BAZ2B (WT) or BAZ2B with the regulatory region removed (10M+) were titrated with unmodified histone 3 (H3) or H3 that was acetylated at lysine-14 (H3K14ac) . In each case, the Hill coefficient was approximately 1.To test binding in a biological setting (in vivo) , WT and 10M+ BAZ2B were extracted from cell lysates on affinity columns containing H3 or H3K14ac covalently attached to beads. BAZ2B was then eluted and binding was quantified by Western blot (Figure 3) .
Figure 1 Electrostatic interactions between H3 and BAZ2B amino acid residues
Figure 2 ITC traces (A) and K_d values (B) of WT BAZ2B and 10M+ BAZ2B titrated with either unmodified H3 or H3Ka4ac
Figure 3 Western blot of BAZ2B recovered from unmodified or acetylated H3 columns
Adapted from Kostrhon S, Kontaxis G, Kaufmann T, et al. A histone-mimicking interdomain linker in a multidomain protein modulates multivalent histone binding. J Biol Chem. 2017;292(43) :17643-17657.
-Which of the proteins mentioned in the passage has the highest isoelectric point?

Question

Passage
Interactions between positively charged histones and negatively charged nucleic acids are altered by acetylation and methylation of histone subunits. These different post-translational modifications are recognized by histone-binding proteins such as BROMO domain Adjacent to Zinc finger 2B (BAZ2B) . BAZ2B has a net negative charge but contains a lysine-rich regulatory region between its two histone-binding domains.NMR data revealed potential interactions between BAZ2B and histone subunit H3 (Figure 1) . Binding was confirmed by isothermal titration calorimetry (ITC) , which measures enthalpy of binding when a protein solution is titrated with ligand injections of equal volume and concentration. Each injection results in a negative peak as heat is released. Figure 2 shows ITC results when wild-type BAZ2B (WT) or BAZ2B with the regulatory region removed (10M+) were titrated with unmodified histone 3 (H3) or H3 that was acetylated at lysine-14 (H3K14ac) . In each case, the Hill coefficient was approximately 1.To test binding in a biological setting (in vivo) , WT and 10M+ BAZ2B were extracted from cell lysates on affinity columns containing H3 or H3K14ac covalently attached to beads. BAZ2B was then eluted and binding was quantified by Western blot (Figure 3) .

Figure 1 Electrostatic interactions between H3 and BAZ2B amino acid residues

Figure 2 ITC traces (A) and K_d values (B) of WT BAZ2B and 10M+ BAZ2B titrated with either unmodified H3 or H3Ka4ac

Figure 3 Western blot of BAZ2B recovered from unmodified or acetylated H3 columns
Adapted from Kostrhon S, Kontaxis G, Kaufmann T, et al. A histone-mimicking interdomain linker in a multidomain protein modulates multivalent histone binding. J Biol Chem. 2017;292(43) :17643-17657.
-Which of the proteins mentioned in the passage has the highest isoelectric point?

Quizplus · Accepted Answer

11ecba2d_0eb0_e060_a3bf_4b465bdb3a5e_MD0008_00 The isoelectric point (pI) of a protein is the pH at which it has a net charge of zero. When the pI is reached, individual residues may still be positively or negatively charged, but there must be an equal number of positive and negative charges so that they cancel each other. A protein with a net positive charge at physiological pH has a high pI because a high pH is needed to deprotonate enough of the positively charged side chains. Conversely, proteins with a net negative charge at physiological pH have a low pI and require a low pH for neutralization. According to the passage, BAZ2B has a net negative charge whereas histone subunits are positively charged at physiological pH. Therefore, both forms of H3 have a higher pI than either form of BAZ2B. According to the passage, H3K14ac is acetylated at a lysine residue. Histone acetylation neutralizes the positive charge of lysine, so H3K14ac has one fewer positive charge than unmodified H3. The pI of H3K14ac is decreased relative to the pI of unmodified H3, so H3 has the highest pI of the proteins listed. (Choices A and B) WT BAZ2B and 10M+ BAZ2B have net negative charges at physiological pH, so they have a lower pI than the histone proteins that are positively charged at physiological pH. (Choice D) H3K14ac is less positively charged than unmodified H3 at physiological pH, so it does not have the highest pI. Educational objective: The isoelectric point (pI) of a protein is the pH at which the protein has a net charge of zero. The more positively charged the protein is at physiological pH, the higher its pI.

Passage Interactions Between Positively Charged Histones and Negatively Charged Nucleic Acids