The effect of stabilizing additives on the structure and hydration of proteins: a study involving tetragonal lysozyme

In order to elucidate the effect of stabilizing additives on the structure of proteins and the associated ordered water molecules in the hydration shell, the crystal structures of tetragonal lysozyme grown in the presence of sucrose, sorbitol and trehalose have been refined. Also refined are the structures of orthorhombic and monoclinic lysozyme grown under the conditions in which tetragonal lysozyme is normally grown. A comparison of the two sets of structures with the structure of native tetragonal lysozyme shows that the effect of the additives on the structure of the protein molecule is less than that of the normal minor changes associated with differences in molecular packing. Surprisingly, the same is true of the effect on the hydration shell, represented by the ordered water molecules attached to the protein. Thus, it appears that the cause of the stabilizing effect of the additives needs to be sought outside the immediate neighbourhood of the protein molecule. Sorbitol and trehalose do not coherently interact with the protein. One sucrose molecule binds at the active-site cleft of the enzyme