Destabilization of the D2 domain of Thermotoga maritima arginine binding protein induced by guanidinium thiocyanate and its counteraction by stabilizing agents

Abstract

D2 is a structural and cooperative domain of Thermotoga maritima Arginine Binding Protein, that possesses a remarkable conformational stability, with a denaturation temperature of 102.6°C, at pH 7.4. The addition of potassium thiocyanate causes a significant decrease in the D2 denaturation temperature. The interactions of thiocyanate ions with D2 have been studied by means of isothermal titration calorimetry measurements and molecular dynamics simulations. It emerged that: (a) 20-30 thiocyanate ions interact with the D2 surface and are present in its first solvation shell; (b) each of them makes several contacts with protein groups, both polar and nonpolar ones. The addition of guanidinium thiocyanate causes a marked destabilization of the D2 native state, because both the ions are denaturing agents. However, on adding to the solution containing D2 and guanidinium thiocyanate a stabilizing agent, such as TMAO, sucrose or sodium sulfate, a significant increase in denaturation temperature occurs. The present results confirm that counteraction is a general phenomenon for globular proteins.

Keywords: Guanidinium; MD simulations; TmArgBP; calorimetry; thermodynamics; thiocyanate.

FIGURE 1

DSC traces of D2 in aqueous 20 mM phosphate buffer, pH 7.4, and in the presence of different KSCN concentrations (a); DSC profiles of the first heating (black solid line) and second heating (red dashed line) of D2 in aqueous buffer solution (b).

FIGURE 2

ITC trace obtained from the titration of a D2 solution (42 μM) with a solution of KSCN 300 mM; the black line is the raw ITC trace obtained from the binding experiment; the red line is the raw ITC trace obtained from the KSCN dilution experiment (a); binding isotherm for the interaction between SCN⁻ and D2: The black squares represent the cumulative heats of interaction obtained from the ITC traces reported in panel a; the red line is the best fit of experimental data according to an independent and equivalent binding site model with a stoichiometry n = 30 ± 10. The experiment was performed in duplicate at the temperature of 25°C in 20 mM phosphate buffer, pH 7.4 (b).

FIGURE 3

Percentage fraction of frames in which a given number of SCN⁻ ions occurs within a shell of 4 Å thickness around the protein surface at 300, 350, and 400 K, respectively, during the entire simulation time (i.e., 1 μs by considering all the 5 MD trajectories performed at each temperature, 10,000 analyzed frames). The obtained mean values are 21.1, 19.9, and 19.0 passing from 300 to 350 K and 400 K, respectively.

FIGURE 4

Minimal distance distribution between thiocyanate atoms and D2 surface atoms. S and N distances are reported at 300, 350, and 400 K. Protein hydrogens are not considered in the calculation.

FIGURE 5

Example of a persistent SCN⁻—binding site in D2: Ghost surface representation is used to localize the binding pocket onto the protein, while ball‐and‐sticks are used for the SCN⁻ ion; SCN⁻ poses, corresponding to different time frames from a single MD run, are reported (a and b); close‐up view of the binding site: Amino acid side chains that stabilize the SCN⁻ ion are labeled and displayed in sticks; spheres are used for SCN⁻ (S = yellow, C = green, N = blue) and the protein is rendered in green cartoons (c).

FIGURE 6

Emergence of a “new” SCN⁻ binding site during the MD trajectories at 400 K; two different orientations are provided. Amino acid side chains that stabilize the SCN⁻ ion are labeled and displayed in sticks; spheres are used for SCN⁻ (S = brown, C = yellow, N = blue); the protein is rendered in yellow cartoons.

FIGURE 7

DSC traces of D2 in aqueous 20 mM phosphate buffer, pH 7.4, in the presence of different GdmSCN concentrations (a); DSC traces of the first heating (black solid line) and second heating (red dashed line) for D2 in buffer solution containing 1, 1.5 and 2 M GdmSCN (b).

FIGURE 8

Percentage fraction of frames in which a given number of SCN⁻ ions or of Gdm⁺ ions occurs within a shell of 4 Å thickness around the protein surface at 300 K, during the entire simulation time (i.e., 1 μs by concatenating the 5 MD trajectories, 10,000 analyzed frames). The obtained mean values are 57.4 for SCN⁻ ions and 65.6 Gdm⁺ ions.

FIGURE 9

DSC traces of D2 in aqueous 20 mM phosphate buffer, pH 7.4, and in the presence of 1 M GdmSCN and 1 M of different cosolutes (a); DSC traces of D2 in aqueous 20 mM phosphate buffer, pH 7.4, and in the presence of 1 M GdmSCN and different concentrations of sodium sulfate (b).