STRUCTURAL AND MORPHOLOGICAL CHARACTERIZATION OF ULTRALENTE INSULIN CRYSTALS BY ATOMIC-FORCE MICROSCOPY - EVIDENCE OF HYDROPHOBICALLY DRIVEN ASSEMBLY

Although x-ray crystal structures exist for many forms of insulin, the hormone involved in glucose metabolism and used in the treatment of d iabetes, x-ray structural characterization of therapeutically importan t long-acting crystalline ultralente insulin forms has been elusive be cause of small crystal size and poor diffraction characteristics. We d escribe tapping-mode atomic force microscopy (TMAFM) studies, performe d directly in crystallization liquor, of ultralente crystals prepared from bovine, human, and porcine insulins. Lattice images obtained from direct imaging of crystal planes are consistent with R3 space group s ymmetry for each insulin type, but the morphology of the human and por cine crystals observed by AFM differs substantially from that of the b ovine insulin crystals. Human and porcine ultralente crystals exhibite d large, molecularly flat (001) faces consisting of hexagonal arrays o f close packed hexamers, In contrast, bovine ultralente crystals predo minantly exhibited faces with cylindrical features assignable to close -packed stacks of insulin hexamers laying in-plane, consistent with th e packing motif of the (010) and (011) planes. This behavior is attrib uted to a twofold increase in the hydrophobic character of the upper a nd lower surfaces of the donut-shaped insulin hexamer in bovine insuli n compared to its human and porcine counterparts that results from min or sequence differences between these insulins. The increased hydropho bicity of these surfaces can promote hexamer-hexamer stacking in precr ystalline aggregates or enhance attachment of single hexamers along th e c axis at the crystal surface during crystal growth. Both events lea d to enhanced growth of {hk0} planes instead of (001). The insulin hex amers on the (010) and (110) faces are exposed ''edge-on'' to the aque ous medium, such that solvent access to the center of the hexamer and to solvent channels is reduced compared to the (001) surface, consiste nt with the slower dissolution and reputed unique basal activity of bo vine ultralente insulin. These observations demonstrate that subtle va riations in amino acid sequence can dramatically affect the interfacia l structure of crystalline proteins.