ATOMIC-FORCE MICROSCOPY OF INSULIN SINGLE-CRYSTALS - DIRECT VISUALIZATION OF MOLECULES AND CRYSTAL-GROWTH

Atomic force microscopy performed on single crystals of three differen t polymorphs of bovine insulin revealed molecularly smooth (001) layer s separated by steps whose heights reflect the dimensions of a single insulin hexamer. Whereas contact mode imaging caused etching that prev ented molecular-scale resolution, tapping mode imaging in solution pro vided molecular-scale contrast that enabled determination of lattice p arameters and polymorph identification while simultaneously enabling r eal-time examination of growth modes and assessment of crystal quality . Crystallization proceeds layer by layer, a process in which the prot ein molecules assemble homoepitaxially with nearly perfect orientation al and translational commensurism. Tapping mode imaging also revealed insulin aggregates attached to the (001) faces, their incorporation in to growing terraces, and their role in defect formation. These observa tions demonstrate that tapping mode imaging is ideal for real-time in situ investigation of the crystallization of soft protein crystals of relatively small proteins such as insulin, which cannot withstand the lateral shear forces exerted by the scanning probe in conventional ima ging modes.