Determination of liposome size: A tool for protein reconstitution Aleksandar Vojta a , Johannes Scheuring b , Nikolaus Neumaier b , Oliver Mirus a , Sevil Weinkauf b , Enrico Schleiff a, * a Department of Biology I, Botany III, Ludwig Maximilians University (LMU) Munich, 80638 Munich, Germany b Department of Chemistry, Technical University (TU) Munich, 85748 Garching, Germany Received 30 March 2005 Available online 26 September 2005 Abstract Reconstitution of proteins into liposomes is a widespread approach to analyzing their biological function. Many protocols exist for this procedure and for the subsequent analysis of proteins. Here, we establish a procedure for preparation and analysis of liposomes with a lipid composition reflecting the outer envelope of chloroplasts. First, the stability of the liposomes in different buffer systems was inves- tigated to provide information for the storage of the reconstituted system. Then, the size of the liposomes created by filtration through a polycarbonate filter dependent on the lipid composition was analyzed. Subsequently, solubilization of the liposomes composed of lipids with the outer envelope composition by dodecylmaltoside and octylglucoside as a preceding step of reconstitution was studied. Finally, we developed a straightforward method to determine the size of liposomes by absorption spectroscopy. The described setup allows the construction of reconstitution protocols, including the final determination of the liposome size. Ó 2005 Elsevier Inc. All rights reserved. Keywords: Liposome size; Liposome stability; Absorption spectroscopy; Light scattering Reconstitution of membrane proteins into synthetic lip- osomes is one of the major tools used to investigate their function, and various reconstitution protocols have been established in the past [1–4]. However, it became obvious that the liposome behavior and architecture depend on the lipid composition. For example, the influence of salt on the liposome structure was found to be lipid dependent [5–7]. In addition, non-bilayer lipids can influence the lipo- some shape during reconstitution or subsequent experi- ments [8,9]. To avoid such influences, many experiments were performed using model lipids such as phosphatidyl- choline. However, recently accumulated evidence shows that the activity of proteins in the bilayer [10–12] and the dynamic complex assembly [13] are altered by the lipid con- tent. Therefore, it is essential to construct reconstitution systems with authentic lipid content. We are interested in the function of the proteins of the chloroplast envelope membranes. On the outer chloroplast envelope, the Toc complex facilitates import of the proteins synthesized in the cytosol into chloroplasts. A reconstituted system was recently used to study the function of this com- plex [4,14]. In that study, the lipid composition of the outer envelope membrane (spinach) was used to create liposomes [15]. For a better understanding of the translocation path- way, it is also necessary to reconstitute other homologs of the translocation components as well as the isolated Tic complex [16]. However, to define efficient reconstitution protocols, the following issues and questions must be ad- dressed. First, liposomes are constructed in different buffer solutions and used for activity assays. How stable are such liposomes against lysis? Second, all liposomes are created by extrusion through a polycarbonate filter. Using the lipid composition of the outer and inner envelope, does this pro- cedure allow the incorporation of all lipids into the lipo- somes? Third, using the polycarbonate filter, the liposome size can theoretically be defined by the pore size of the fil- ter. Can this size dependence be confirmed and controlled? 0003-2697/$ - see front matter Ó 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.ab.2005.09.003 * Corresponding author. Fax: +49 89 17861 185. E-mail address: schleiff@lrz.uni-muenchen.de (E. Schleiff). www.elsevier.com/locate/yabio Analytical Biochemistry 347 (2005) 24–33 ANALYTICAL BIOCHEMISTRY