Designer Histone Complexes: Controlling Protein–DNA Interactions with Protein Charge as an “All-or-None” Digital Switch

Abstract

An artificial histone is synthesized that functions as a DNA-protein digital switch, where DNA binding is all or none, controlled by a sharp threshold of protein charge. A non-DNA-binding protein, glucose oxidase (GOx), was chemically modified by attaching an increasing number of triethylenetetramine (TETA) side chains to its glutamate/aspartate groups to obtain a small library of covalently modified GOx(n) derivatives. The parameter n denotes the net charge on the protein at pH 7, which was increased from -62 (pristine GOx) to +75 by attaching an increasing number of TETA residues to the protein. All GOx(n) derivatives retained their secondary structure to a good extent, as monitored by UV circular dichroism (CD) spectroscopy, and they also retained oxidase activities to a significant extent. The interaction of the GOx(n) with calf thymus DNA was examined by isothermal titration calorimetry (ITC). Pristine GOx of -62 charge at pH 7 in 10 mM Tris-HCl and 50 mM NaCl buffer had no affinity for the negatively charged DNA helix, and GOx(n) with n < +30 had no affinity for DNA either. However, binding has been turned on abruptly when n ≥ +30 with binding constants (K) ranging from (1.5 ± 0.7) × 10 to (7.3 ± 2.8) × 10 M for n values of +30 and +75, respectively, and this type of "all-or-none" binding based on protein charge is intriguing. Furthermore, thermodynamic analysis of the titration data revealed that binding is entirely entropy-driven with ΔS ranging from 0.09 ± 0.007 to 0.19 ± 0.008 kcal/mol K with enthalpic penalties of 17.0 ± 2.3 and 46.1 ± 2.1 kcal/mol, respectively. The binding had intrinsic propensities (ΔG) ranging from -9.8 ± 0.14 to -10.7 ± 0.25 kcal/mol, independent of n. DNA binding distorted protein-DNA secondary structure, as evidenced by CD spectroscopy, but oxidase activity of GOx(n)/DNA complexes has been unaffected. This is the very first example of an artificial histone (GOx(n)) where the protein charge functioned as a DNA-binding switch; protein charge is in turn under complete chemical control while preserving the biological activity of the protein. The new insight gained here could be useful in the design of novel "on-off" protein switches.

Tuning Enzyme/α-Zr(IV) Phosphate Nanoplate Interactions via Chemical Modification of Glucose Oxidase

“…A peak shift from 142,896.3 Da observed for pristine GOx to 149,344.3 Da for the modified GOx indicated a total number of 44 TETA side chains per GOx( n ), and had a net charge of +75. 43 Therefore, MS indicated a difference of +5 charge units when compared to electrophoresis methods. Thus, the error associated with the quantification of the number of net charge units by the agarose could be as much as ±5 units.…”

“…The charge of GOx­(+70) assigned by the gel electrophoresis method was also examined by mass spectrometry and m / z analysis. A peak shift from 142,896.3 Da observed for pristine GOx to 149,344.3 Da for the modified GOx indicated a total number of 44 TETA side chains per GOx­( n ), and had a net charge of +75 . Therefore, MS indicated a difference of +5 charge units when compared to electrophoresis methods.…”

“…We recently reported a charge threshold of +30 for the binding of GOx( n ) to double helical DNA. 43 However, there are many differences between the two, and one is that GOx( n )/ α -ZrP interaction is observed even when n = −20. However, there is no observable GOx( n )/DNA interaction below n = +30.…”

“…In fact, these data indicate a threshold number of amines below which there is Δ G charge dependence and above which other factors make a significant contribution to the binding thermodynamics. We recently reported a charge threshold of +30 for the binding of GOx­( n ) to double helical DNA . However, there are many differences between the two, and one is that GOx­( n )/α-ZrP interaction is observed even when n = −20.…”