Proteoliposomes are systems that mimic lipid membranes (liposomes) to which a protein has been incorporated or inserted. During the last decade, these systems have gained prominence as tools for biophysical studies on lipid-protein interactions as well as for their biotechnological applications. Proteoliposomes have a major advantage when compared with natural membrane systems, since they can be obtained with a smaller number of lipidic (and protein) components, facilitating the design and interpretation of certain experiments. However, they have the disadvantage of requiring methodological standardization for incorporation of each specific protein, and the need to verify that the reconstitution procedure has yielded the correct orientation of the protein in the proteoliposome system with recovery of its functional activity. In this review, we chose two proteins under study in our laboratory to exemplify the steps necessary for the standardization of the reconstitution of membrane proteins in liposome systems: (1) alkaline phosphatase, a protein with a glycosylphosphatidylinositol anchor, and (2) Na,K-ATPase, an integral membrane protein. In these examples, we focus on the production of the specific proteoliposomes, as well as on their biochemical and biophysical characterization, with emphasis on studies of lipid-protein interactions. We conclude the chapter by highlighting current prospects of this technology for biotechnological applications, including the construction of nanosensors and of a multiprotein nanovesicular biomimetic to study the processes of initiation of skeletal mineralization.

Globular proteins composed of different secondary structures and fold types were examined by synchrotron radiation circular dichroism spectroscopy to determine the effects of dehydration on their secondary structures. They exhibited only minor changes upon removal of bulk water during film formation, contrary to previously reported studies of proteins dehydrated by lyophilization (where substantial loss of helical structure and gain in sheet structure was detected). This near lack of conformational change observed for globular proteins contrasts with intrinsically disordered proteins (IDPs) dried in the same manner: the IDPs, which have almost completely unordered structures in solution, exhibited increased amounts of regular (mostly helical) secondary structures when dehydrated, suggesting formation of new intra‐protein hydrogen bonds replacing solvent‐protein hydrogen bonds, in a process which may mimic interactions that occur when IDPs bind to partner molecules. This study has thus shown that the secondary structures of globular and intrinsically disordered proteins behave very differently upon dehydration, and that films are a potentially useful format for examining dehydrated soluble proteins and assessing IDPs structures.