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Binding Affinity and Function of the Extremely Disordered Protein Complex Containing Human Linker Histone H1.0 and Its Chaperone ProTα - PubMed

  • ️Mon Jan 01 2018

Binding Affinity and Function of the Extremely Disordered Protein Complex Containing Human Linker Histone H1.0 and Its Chaperone ProTα

Hanqiao Feng et al. Biochemistry. 2018.

Abstract

It was recently reported that human linker histone H1.0 and its chaperone prothymosin-α (ProTα) form an extremely disordered 1:1 complex with an ultrahigh affinity (equilibrium dissociation constant KD of ∼2 × 10-12 M) measured using a single-molecule Förster resonance energy transfer method. It was hypothesized that the ultrahigh affinity and extreme disorder may be required for the chaperone function of ProTα, in which it displaces the linker histone from condensed chromatin. Here, we measure the binding affinity for the ProTα-H1.0 complex using isothermal titration calorimetry and report a KD value of (4.6 ± 0.5) × 10-7 M. In addition, we show that ProTα facilitates the formation of the H1.0-nucleosome complex in vitro. The results of our study contrast with those of the previous report and provide new insights into the chaperone function of ProTα. Possible causes for the observed discrepancy in binding affinity are discussed.

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Figures

Figure 1.
Figure 1.

Binding affinity of H1.0 and ProTα measured using ITC. (a) Illustration of a representative titration profile at an ionic strength (IS) of 165 mM. The filled circles represent the experimental data. The solid line is the fitting curve. (b) Comparison of the ITC results (●) with those measured using the smFRET method (□). The solid lines are the fitting curves: a linear function to the ITC data and an exponential function to the smFRET data. The table shows the ionic strengths and measured KD values from the ITC experiments. The KD value is the average of the results from two ITC experiments. The uncertainty shows the deviation from the average.

Figure 2.
Figure 2.

ProTα facilitates the formation of the H1.0–nucleosome complex. (a) A 5% native polyacrylamide gel electrophoresis EMSA on the free nucleosome, H1.0, and ProTα mixed at the indicated ratios. (b) Plots of the integrated intensities within the corresponding rectangular boxes (marked with i–iii) on the gel. Note that the nucleosome–H1.0 complex is more compact than the nucleosome and runs faster in the gel.

Figure 3.
Figure 3.

Size-exclusion experiments showed no formation of higher-order oligomers when H1.0 is in excess. Elution profiles of the ProTα–H1.0 complex (black solid line) at concentration of ∼10 μM for each of the proteins and the same complex with additional amounts of H1.0.

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