Study of Receptor-Chaperone Interactions Using the Optical Technique of Spectroscopic Ellipsometry

Abstract

This work describes a detailed quantitative interaction study between the novel plastidial chaperone receptor OEP61 and isoforms of the chaperone types Hsp70 and Hsp90 using the optical method of total internal reflection ellipsometry (TIRE). The receptor OEP61 was electrostatically immobilized on a gold surface via an intermediate layer of polycations. The TIRE measurements allowed the evaluation of thickness changes in the adsorbed molecular layers as a result of chaperone binding to receptor proteins. Hsp70 chaperone isoforms but not Hsp90 were shown to be capable of binding OEP61. Dynamic TIRE measurements were carried out to evaluate the affinity constants of the above reactions and resulted in clear discrimination between specific and nonspecific binding of chaperones as well as differences in binding properties between the highly similar Hsp70 isoforms.

Yeast Cytochrome c Monolayer on Flat and Nanostructured Gold Films Studied by UV–Vis Spectroscopic Ellipsometry

“…Spectroscopic Ellipsometry Ellipsometry and UV-vis SE are often used to investigate adsorption dynamics at surfaces and thickness of thin and ultrathin biological films [22][23][24][25][26] and other complex molecular systems [7,8,[27][28][29][30][31].…”

Analysis of Toxic Amyloid Fibril Interactions at Natively Derived Membranes by Ellipsometry

“…Cell deposition and TIRE analysis were performed as previously described [ 24 – 25 ]. Briefly, Langmuir-Schaefer deposition was performed using standard microscopic glass sides (1" x 1") coated with chromium (Cr 3nm thick) and gold (Au 25 nm thick) via thermal evaporation (Edwards Life Sciences A360 metallizing unit, Irvine, CA).…”

“…During sample incubation, two types of ellipsometric measurements were performed: (1) dynamic TIRE spectral measurements where multiple TIRE spectra were recorded during protein adsorption; and (2) TIRE single spectrum scan recording after completion of every adsorption step using standard buffer solution (1 x PBS, pH 8.0). Software provided by J.A Woollam Ltd enabled the modelling of the reflection system and subsequent evaluation of the thickness and refractive index of adsorbed molecular layers, comparing experimental and theoretical values of Ψ and Δ (previously described [ 24 ]). A single spectroscopic scan performed after completion of sample incubation per concentration enabled the recording and interpretation of direct changes to membrane thickness (Δd, nm).…”

“…Our methodology allows the deposition of whole organelles or cells onto a gold coated surface, resulting in a natively derived membrane on which we can perform interaction studies. Previously, we used this method to study protein receptor interactions on both chloroplasts [ 24 ] and plasma membranes [ 25 ], indicating that the deposited membrane remains largely intact and is capable of binding to soluble proteins in a specific manner. Here we analyze unfragmented and fragmented amyloid fibril interactions with natively derived cellular membranes.…”

Analysis of Protein Interactions at Native Chloroplast Membranes by Ellipsometry

“…TIRE has been successfully applied to analyze interactions between antibodies and various ligands [3] , including the analysis of low molecular weight analytes such as pesticides [5] and mycotoxins with detection levels as low as 0.1 ng/ml [6] . TIRE has also been used to determine the binding affinity of the molecular chaperone Hsp70 for a soluble form of its receptor OEP61 bound to a gold surface, and was sensitive enough to discriminate between the binding affinities of closely related Hsp70 isoforms [7] , [8] . This well characterized interaction [7] between Hsp70 and OEP61 is exploited here to measure specific protein-protein interactions at a native membrane.…”