In Situ Determination of Molecular Packing and Growth Mechanisms in Protein Crystals: Atomic Force Microscopy of Insulin and Insulin Analogs. Christopher M. Yip¹, Michael R. DeFelippis¹, Michael D. Ward², Mark L. Brader¹, ¹Lilly Research Laboratories, Eli Lilly and Company, Indianapolis, IN 46285, ²Department of Chemical Engineering and Materials Science and the Center for Interfacial Engineering, University of Minnesota, Minneapolis, MN 55455

The treatment of diabetes relies on subcutaneous injections of either freely soluble or microcrystalline suspension forms of insulins. However, the small dimensions of the microcrystalline forms precludes facile analysis by conventional diffraction techniques. The relationship between the crystal dissolution kinetics and the pharmocokinetics of the insulin therapy also mandates an understanding of the molecular packing of individual insulin hexamers and local disorder at the crystal-solution interface. Recent advances in scanning probe microscopy provide a means of addressing these issues.

Real-time in situ imaging of insulin and insulin analog crystal growth by tapping mode AFM revealed the formation of voids and solvent cores through attachment of misaligned aggregates. Furthermore, a comparison of the dislocation morphologies and growth modes suggested that subtle changes in the free energy of kink addition and the interfacial surface energy of the growing crystal step are responsible for variations in crystal morphology. In situ high resolution AFM performed on cubic wild-type insulin crystals grown from high salt solutions revealed two-dimensional molecular scale periodicities consistent with the packing motif of individual insulin hexamers. The surface periodicities and lattice orientations were found to coincide with the external morphology of individual crystal facets. Reconstruction of the three-dimensional packing arrangement of the insulin hexamers based on molecular modeling of the observed surface periodicities revealed that these crystal forms likely belong to the R3 space group. Our studies demonstrate the application of in situ AFM for the characterization of interfacial structure and dynamics in protein crystal systems, including direct real space visualization of molecular packing, growth events, and identification of space group symmetry.