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

Abstract

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 Lys(B28)Pro(B29) (NPL), a complex 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 intermediate time-action therapy for treating insulin-dependent diabetes. Tapping mode AFM performed at 6 degrees C on bipyramidally tipped tetragonal rod-shaped NPL crystals revealed large micron-sized islands separated by 44-A 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 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 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.

Insulin Analogs ‐ Improving the Therapy of Diabetes

“…The NPH-like crystals of insulin lispro are also oblong tetragonal crystals, similar to those observed with NPH insulin formulations [56]. The NPH-like suspension of insulin lispro possesses the physicochemical properties relative to human insulin NPH [60]; however, to prepare the NPHanalogous tetragonal crystals with intermediate-acting time-action, significant modifications to the protamine/insulin crystallization procedure was required due to the reduced self-association properties of insulin lispro [61].…”

Simultaneous nanoparticle formation and encapsulation driven by hydrophobic interaction of casein-graft-dextran and β-carotene

“…5E and 5F. The height mode of AFM reflects the surface property of the object, while the phase mode measures the stiffness or elasticity difference of the object [32]. For β-carotene/copolymer particles, the height image shows globular shape with smooth surface.…”

Structure and growth of ultrasmall protein microcrystals by synchrotron radiation: II. µGISAX and microscopy of lysozyme

“…Due to the unique combination of GISAXS and micrometer sized beam, our investigation of lysozymes confirms the higher performance of the nanotemplate method in producing crystals and provides new insights on the differential early steps of crystal nucleation and growth within the drop for both the lysosymes and the cytochromes. µGISAXS data allow indeed monitoring crystal formation at a resolution and a scale previously unmatchable even with respect to what has been significantly achieved by AFM studies [Li et al, 1999; Yip et al, 2000; McPherson et al, 2003].…”

“…This technique can be used to measure the growth unit sizes on protein crystal faces as well, allowing their molecular growth mechanisms to be determined experimentally. It was applied to probe the growth mechanism of the (110) face of tetragonal lysozyme crystals [Li et al, 1999]; in this study the latest step of crystal growth is observed, often on the large crystal terrace [Yip et al, 2000].…”