(765a) Cryo-TEM Characterization of Recombinant Protein Self-Assembly Into Sheets, Fibers, and Vesicles

Nanostructures created through the self-assembly of recombinant surfactant proteins allows for the precise control of surfactant chemistry and simplistic functionalization through molecular biology.   We report the formation of self-assembled sheets, fibers, and vesicles from a family of recombinantly-produced oleosin, a naturally occurring plant protein.  Protein mutants were characterized through Western blot analysis, MALDI mass spectroscopy, and circular dichroism.  Solutions of these proteins were injected into various aqueous buffers and structural characterization was completed through cryo-transmission electron microscopy.  The phase behavior of the mutant family was studied in several ionic strength buffers showing that a rich phase behavior could be obtained by changing the salt concentration in solution.  Circular dichroism revealed that the variety of structures seen was not heavily influences by changes in the secondary structure across the family.  In vesicle systems, an increase in hydrophilic fraction at a fixed hydrophobic domain leads to an increase in membrane thickness.   The formation of a bilayer membrane in vesicles was confirmed on the micron scale through duel encapsulation of hydrophilic dye in the lumen and hydrophobic dye in the membrane.  The use of recombinant techniques to create surfactant proteins allows for compete control over surfactant chemistry and molecular weight, a major advantage over previous polydisperse systems.