THE MECHANISM OF PROTEIN CRYSTAL-GROWTH FROM LIPID LAYERS

Two-dimensional (2D) crystals of proteins on lipid monolayers can init iate the formation of large three-dimensional (3D) crystals suitable f or X-ray diffraction studies. The role of the 2D crystals in this proc ess has not been firmly established. While it is likely that the 2D cr ystals serve as nuclei for epitaxial crystal growth, other mechanisms, such as non-specific nucleation induced by the high local concentrati on of the protein at the surface of the Lipid layer, cannot be exclude d. Using streptavidin as a model system, we have now firmly establishe d that 3D crystal growth from 2D crystals on lipid layers occurs by ep itaxy. We show that 2D crystals of streptavidin (space group C222) on biotinated lipid layers nucleate the growth of a 3D crystal form (spac e group I4(I)22) that possesses a structural similarity with the 2D cr ystal, but have no effect on the growth of 3D crystal forms (I222 and P2(1)) that are unrelated to the 2D crystal. At lower pH, a new 3D cry stal form (space group Pi), unrelated to the previously described 2D c rystals, grew from Lipid layers. This discovery initially raised conce rn about the validity of the epitaxial mechanism, but these concerns w ere alleviated with the subsequent discovery of a structurally related 2D P1 crystal that grew in similar solution conditions. Some paramete rs affecting epitaxial growth of both the Pi and I4(I)22 crystals were investigated, revealing several noteworthy features of the epitaxial growth. (1) 2D crystals are very effective nucleating agents; for inst ance, the P1 2D crystals can direct the growth of P1 3D crystals even under conditions that favour the growth of other crystal forms. (2) Th e epitaxial 3D crystal grow very rapidly and at amazingly low protein concentrations; P1 3D crystals can be grown from solutions as low as 1 0 mu M streptavidin. (3) There is no obligate requirement for the depo sition of ; pre-formed 2D crystals; lipid layers alone are equally eff ective at promoting epitaxial crystal growth.