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.
-Based on Figures 2 and 3, which combination of histone 3 (H3 or H3K14ac) and 10M+ BAZ2B has the highest binding affinity in vitro and in vivo?

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.
-Based on Figures 2 and 3, which combination of histone 3 (H3 or H3K14ac) and 10M+ BAZ2B has the highest binding affinity in vitro and in vivo?

Quizplus · Accepted Answer

11ecba2d_0eaa_ecec_a3bf_b18ccc58ce77_MD0008_00 In vitro assays can be used to measure the parameters of purified biomolecules such as proteins. They are tightly controlled experiments performed outside of living organisms that allow variables to be isolated because only a few types of molecules are present. However, in vitro assays are limited in their usefulness because they may not accurately reflect the behavior of a complex biological system presented by in vivo assays, which are conducted in whole living organisms with thousands of different molecules present. Therefore, results from in vitro assays must be confirmed by in vivo assays. The passage illustrates a case in which in vitro and in vivo results do not fully agree. In the ITC experiment (an in vitro technique that uses purified proteins), the dissociation constant K_d is used to determine the binding affinity of proteins. The low K_d of the 10M+ BAZ2B interaction with H3 shows that it has a higher binding affinity with H3 than with H3K14ac. In the in vivo experiment, in which proteins were extracted directly from cells, the results are the opposite. 10M+ BAZ2B has a higher affinity for H3K14ac than it has for unmodified H3, as shown by the thicker band in the H3K14ac lane of the western blot, which indicates increased binding. (Choice B) This option is the opposite of the correct answer. 10M+ BAZ2B binds H3 with the highest affinity in vitro, but H3K14ac in vivo. (Choice C) H3K14ac has the highest 10M+ BAZ2B affinity in vivo, but not in vitro. (Choice D) H3 has the highest 10M+ BAZ2B affinity in vitro, but not in vivo. Educational objective: In vitro assays are useful for their ability to isolate variables, but in vivo assays are required to confirm the behavior of molecules in a biological setting. Binding affinity can be measured by the dissociation constant K_d, where a low K_d corresponds to high binding affinity. It can also be assessed qualitatively by affinity chromatography and western blot, in which a thick, dense band indicates more protein and therefore higher affinity.

Passage Interactions Between Positively Charged Histones and Negatively Charged Nucleic Acids