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Biophys J, January 2000, p. 466-473, Vol. 78, No. 1

Structural Studies of a Crystalline Insulin Analog Complex with Protamine by Atomic Force Microscopy

Christopher M. Yip,* Mark L. Brader,* Bruce H. Frank,* Michael R. DeFelippis,* and Michael D. Warddagger

 *Lilly Research Laboratories, Eli Lilly and Company, Indianapolis, Indiana 46285 and  dagger Department of Chemical Engineering and Materials Science and the Center for Interfacial Engineering, University of Minnesota, Minneapolis, Minnesota 55455 USA

Crystallographic studies of insulin-protamine complexes, such as neutral protamine 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 (TMAFM) studies of crystalline neutral protamine LysB28ProB29 (NPL), a complex of LysB28ProB29 insulin, in which the C-terminal prolyl and lysyl residues of human insulin are inverted, and protamine that is used as an intermediate time-action therapy for treating insulin-dependent diabetes. Tapping mode AFM performed at 6°C on bipyramidally tipped tetragonal rod-shaped NPL crystals revealed large micron-sized islands separated by 44-Å tall steps. Lattice images obtained by in situ TMAFM phase and height imaging on these islands were consistent with the arrangement of individual insulin-protamine complexes on the P41212 (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 height and phase images indicated that tip-sample adhesion was larger in the interstices between NPL complexes in the (110) crystal plane than over the individual complexes. These results demonstrate the utility of low-temperature TMAFM height and phase imaging for the structural characterization of biomolecular complexes.

Biophys J, January 2000, p. 466-473, Vol. 78, No. 1
© 2000 by the Biophysical Society   0006-3495/00/01/466/08  $2.00


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