Structural studies of a crystalline insulin analog complex with protamine by atomic force microscopy

Crystallographic studies of insulin-protamine complexes, such as neutral pr otamine Hagedorn (NPH) insulin, have been hampered by high crystal solvent content, small crystal dimensions, and extensive disorder in the protamine molecules. We report herein in situ tapping mode atomic force microscopy (T MAFM) studies of crystalline neutral protamine Lys(B28)Pro(B29) (NPL), a co mplex of Lys(B28)Pro(B29) insulin, in which the C-terminal prolyl and lysyl residues of human insulin are inverted, and protamine that is used as an i ntermediate time-action therapy for treating insulin-dependent diabetes. Ta pping mode AFM performed at 6 degrees C on bipyramidally tipped tetragonal rod-shaped NPL crystals revealed large micron-sized islands separated by 44 -Angstrom, tall steps. Lattice images obtained by in situ TMAFM phase and h eight imaging on these islands were consistent with the arrangement of indi vidual insulin-protamine complexes on the P4(1)2(1)2 (110) crystal plane of NPH, based on a low-resolution x-ray diffraction structure of NPH, arguing that the NPH and NPL insulins are isostructural. Superposition of the heig ht and phase images indicated that tip-sample adhesion was larger in the in terstices between NPL complexes in the (110) crystal plane than over the in dividual complexes. These results demonstrate the utility of low-temperatur e TMAFM height and phase imaging for the structural characterization of bio molecular complexes.