Influence of Glycan Agents on Protein Crystallization with Ammonium Sulfate

Abstract

Structural information from the high-resolution crystal analysis is advantageous for designing novel pharmaceutics or engineering functional proteins. Obtaining high-quality protein crystals is, however, a hurdle. Ammonium sulfate (AS) is frequently used as a precipitant for modulating the solubility of the protein. The inclusion of glycans in salt-type precipitants is sometimes effective for improving the resolution of X-ray diffractions of protein crystals. In this work, we demonstrate that glycoside-glycan agents improve the quality of crystal growth for two kinds of proteins, influenza virus polymerase acidic subunit Nterminal domain (PA N ) and chitosanase. Molecular dynamics simulations were performed for the model systems consisting of glycans and proteins to clarify their interaction. Several binding poses of glycans on the protein surface were observed in the simulations. Root mean square deviation and the radius of gyration indicated the stability of the glycan binding. A comparison in B-factors between calculation models with and without glycans confirmed the rigidity of the protein conformations at the contact region. Principal component analysis of the simulation trajectories suggested a significantly compact motion of the glycan−protein complex. The binding free energy showed that hydrophobic interactions primarily stabilize the complexes. The relationship between the electrostatic potential around the proteins and glycan binding was closely examined in the simulations. Most of the glycan-binding sites were at the boundary regions between the positive and negative areas of electrostatic potential, where the absolute values of the electrostatic potential were low. The boundary regions coincide with the contact area among proteins in the molecular packing of crystals. Further, the Protein Data Bank was searched for crystal structures obtained by adding glycan agents in AS solution. The information on the inclusion of glycans was still limited in the protein crystallization condition compared to other conventional chemicals. The additives have merits for protein crystallization in increasing protein stability, decreasing complex surface flexibility, and supporting the protein−protein association. The findings of this work will be helpful in making use of glycan agents in protein crystal growth.