Creation of Cross-Linked Crystals With Intermolecular Disulfide Bonds Connecting Symmetry-Related Molecules Allows Retention of Tertiary Structure in Different Solvent Conditions

Abstract

Protein crystals are generally fragile and sensitive to subtle changes such as pH, ionic strength, and/or temperature in their crystallization mother liquor. Here, using T4 phage lysozyme as a model protein, the three-dimensional rigidification of protein crystals was conducted by introducing disulfide cross-links between neighboring molecules in the crystal. The effect of cross-linking on the stability of the crystals was evaluated by microscopic observation and X-ray diffraction. When soaking the obtained cross-linked crystals into a precipitant-free solution, the crystals held their shape without dissolution and diffracted to approximately 1.1 Å resolution, comparable to that of the non-cross-linked crystals. Such cross-linked crystals maintained their diffraction even when immersed in other solutions with pH values from 4 to 10, indicating that the disulfide cross-linking made the packing contacts enforced and resulted in some mechanical strength in response to changes in the preservation conditions. Furthermore, the cross-linked crystals gained stability to permit soaking into solutions containing high concentrations of organic solvents. The results suggest the possibility of obtaining protein crystals for effective drug screening by introducing appropriate cross-linked disulfide bonds.

Changes in advanced protein structure during dense phase carbon dioxide induced gel formation in golden pompano surimi correlate with gel strength

“…Under normal pH of surimi (near neutral), the ionic bond is the main force contributing to the natural structure of myogenic fibronectin. However, the ionic bond between protein molecules can be broken by external factors, such as heat, salt, and high pressure, resulting in protein aggregation and gelation (Hiromoto et al, 2022). As shown in Figure 4, compared with the control group (16.4%), the ionic bond of golden pompano surimi showed different degrees of decrease under different DPCD treatment intensities.…”

Porous protein crystals: synthesis and applications

“…58,59 Oxidation of proximal cysteines to promote the formation of disulfide bonds within LPCs is another well-established strategy. [60][61][62] While sortases [63][64][65] and SpyCatcher/SpyTag 66 have been well studied in the context of enzyme immobilization and installation of guests within or on scaffolds, these systems have been less characterized for their use in stabilizing the scaffold itself.…”