title: Analysis of protein interactions at native chloroplast membranes by ellipsometry.
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 generated using soluble forms of the chloroplast chaperone receptors, OEP61 and Toc64. Therefore, we conclude that chloroplasts have the capacity to selectively bind chaperones, consistent with the notion that chaperones play an important role in protein targeting to chloroplasts. Importantly, this method of monitoring by TIRE does not require any protein labelling. This novel combination of techniques should be applicable to a wide variety of membranes and membrane protein receptors, thus presenting the opportunity to quantify protein interactions involved in fundamental cellular processes, and to screen for drugs that target membrane proteins.
authors.affiliation: Biomedical Research Centre, Sheffield Hallam University, Sheffield, United Kingdom. v.kriechbaumer@shu.ac.uk
authors.forename: Verena
authors.initials: V
authors.lastname: Kriechbaumer
authors.forename: Alexei
authors.initials: A
authors.lastname: Nabok
authors.forename: Mohd K
authors.initials: MK
authors.lastname: Mustafa
authors.forename: Rukaiah
authors.initials: R
authors.lastname: Al-Ammar
authors.forename: Anna
authors.initials: A
authors.lastname: Tsargorodskaya
authors.forename: David P
authors.initials: DP
authors.lastname: Smith
authors.forename: Ben M
authors.initials: BM
authors.lastname: Abell
mesh_terms: D002736:Chloroplasts / Q000378:metabolism*; D018840:HSP70 Heat-Shock Proteins / Q000378:metabolism; D018841:HSP90 Heat-Shock Proteins / Q000378:metabolism; D018532:Pisum sativum / Q000378:metabolism*; D010940:Plant Proteins / Q000378:metabolism*; D025941:Protein Interaction Mapping / Q000379:methods*
chemical_list: D018840:HSP70 Heat-Shock Proteins; D018841:HSP90 Heat-Shock Proteins; D010940:Plant Proteins