Significant role of electrostatic interactions for stabilization of protein assemblies.
Contribution of electrostatic interactions to stability of BPTI orthorhombic, pig-insulin cubic crystals, and horse L ferritin crystals was evaluated with numerical calculation of Poisson-Boltzmann equation based on a dielectric model. The stability of a ferritin molecule (24-mer) composed of 24 subunits was also evaluated. It was found that the surface charge-charge interactions at separation distances (< 5 A) were insensitive to variations in the ionic strength, and thus stabilized assembled states of the proteins (i.e., crystalline state and oligomeric state). It was also revealed that the charge density and the packing of the protein crystals were largely responsible for the ionic strength dependence of the crystal stability. The stability of the 5PTI crystalline state with a high charge density drastically increased as the concentration of the solvent ions increased. In contrast, that of the insulin crystal with a low charge density and large solvent region was insensitive to changes in the ionic concentration. The electrostatic interaction between ferritin 24-mers was attributed to two salt bridges mediated by Cd ion. For the stability of the ferritin 24-mer, which is evolutionally designed, the electrostatic stabilization between the subunits was attributed to polar bonds such as buried salt bridges or hydrogen bonds, which occasionally yielded more than 5 kcal/mol and were numerous and very strong compared with the bonds between molecules in the 5PTI and 9INS crystals. By analyzing the atomic charge-charge interactions in detail, it was found that charge pairs separated by less than 3 A, such as hydrogen bonds, dominantly stabilize the assembled states, and that pairs 3 to 5 A apart were also important. The stability of the assembled states evaluated by the total EET was determined by the fine balance between the two competing contributions arising from the stabilizing atoms and the destabilizing atoms. Changes of the ASA and hydration free energy were also evaluated in accordance with the process of the subunit assembly. The change of hydration free energy, which was very large (i.e., approximately +100 kcal/mol/subunit) and unfavorable for the assembly, was proportional to the electrostatic hydration energy (i.e., Born energy change in the hydration process). Hydrophobic groups were likely to appear more frequently than hydrophilic groups at the interfaces. This study offers a method which can improve the stability of protein crystals by introducing polar or charged residues that are properly designed to form specific hydrogen bonds or salt bridges between neighboring protein molecules. This method is also applicable to crystallography, because it improves refinement of protein structures in crystals by taking the inter-protein interactions into account.
Takahashi
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Department of Life Sciences, Graduate School of Arts and Sciences, University of Tokyo, Japan.
D000465:Algorithms; D003460:Crystallization; D004355:Drug Stability; D008024:Ligands; D008956:Models, Chemical; D011506:Proteins / Q000737:chemistry*; D055672:Static Electricity; D013816:Thermodynamics
D008024:Ligands; D011506:Proteins