The Mechanism of Protein Crystal Growth from Lipid Layers

Abstract

Two-dimensional (2D) crystals of proteins on lipid monolayers can initiate the formation of large three-dimensional (3D) crystals suitable for X-ray diffraction studies. The role of the 2D crystals in this process has not been firmly established. While it is likely that the 2D crystals serve as nuclei for epitaxial crystal growth, other mechanisms, such as non-specific nucleation induced by the high local concentration of the protein at the surface of the lipid layer, cannot be excluded. Using streptavidin as a model system, we have now firmly established that 3D crystal growth from 2D crystals on lipid layers occurs by epitaxy. We show that 2D crystals of streptavidin (space groupC222) on biotinated lipid layers nucleate the growth of a 3D crystal form (space groupI4I22) that possesses a structural similarity with the 2D crystal, but have no effrect on the growth of 3D crystal forms (I222 andP21) that are unrelated to the 2D crystal. At lower pH, a new 3D crystal form (space groupP1), unrelated to the previously described 2D crystals, grew from lipid layers. This discovery initially raised concern about the validity of the epitaxial mechanism, but these concerns were alleviated with the subsequent discovery of a structurally related 2DP1 crystal that grew in similar solution conditions. Some parameters affecting epitaxial growth of both the P1 andI4I22 crystals were investigated, revealing several noteworthy features of the epitaxial growth. (1) 2D crystals are very effective nucleating agents; for instance, theP1 2D crystals can direct the growth ofP1 3D crystals even under conditions that favour the growth of other crystal forms. (2) The epitaxial 3D crystal grow very rapidly and at amazingly low protein concentrations;P1 3D crystals can be grown from solutions as low as 10μM streptavidin. (3) There is no obligate requirement for the deposition of pre-formed 2D crystals; lipid layers alone are equally effective at promoting epitaxial crystal growth.