analysis of protein interactions at native chloroplast membranes by ellipsometry分析蛋白质相互作用在本地叶绿体膜由椭圆光度法.pdfVIP

Analysis of Protein Interactions at Native Chloroplast Membranes by Ellipsometry 1 2 2 2 2 Verena Kriechbaumer *, Alexei Nabok , Mohd K. Mustafa , Rukaiah Al-Ammar , Anna Tsargorodskaya , David P. Smith1, Ben M. Abell1 1 Biomedical Research Centre, Sheffield Hallam University, Sheffield, United Kingdom, 2 Materials and Engineering Research Institute, Sheffield Hallam University, Sheffield, United Kingdom Abstract Membrane bound receptors play vital roles in cell signaling, and are the target for many drugs, yet their interactions with ligands are difficult to study by conventional techniques due to the technical difficulty of monitoring these interactions in lipid environments. In particular, the ability to analyse the behaviour of membrane proteins in their native membrane environment is limited. Here, we have developed a quantitative approach to detect specific interactions between low- abundance chaperone receptors within native chloroplast membranes and their soluble chaperone partners. Langmuir- Schaefer film deposition was used to deposit native chloroplasts onto gold-coated glass slides, and interactions between the molecular chaperones Hsp70 and Hsp90 and their receptors in the chloroplast membranes were detected and quantified by total internal reflection ellipsometry (TIRE). We show that native chloroplast membranes deposited on gold- coated glass slides using Langmuir-Schaefer films retain functional receptors capable of binding chaperones with high specificity and affinity. Taking into account the low chaperone receptor abundance in native membranes, these binding properties are consistent with data