Reconstitution of Functional Integrin αIIbβ3 and Its Activation in Plasma Membrane-Mimetic Lipid Environments

Abstract

The study of the platelet receptor integrin αIIbβ3 in a membrane-mimetic environment without interfering signalling pathways is crucial to understand protein structure and dynamics. Our understanding of this receptor and its sequential activation steps has been tremendously progressing using structural and reconstitution approaches in model membranes, such as liposomes or supported-lipid bilayers. For most αIIbβ3 reconstitution approaches, saturated short-chain lipids have been used, which is not reflecting the native platelet cell membrane composition. We report here on the reconstitution of label-free full-length αIIbβ3 in liposomes containing cholesterol, sphingomyelin, and unsaturated phosphatidylcholine mimicking the plasma membrane that formed supported-lipid bilayers for quartz-crystal microbalance with dissipation (QCM-D) experiments. We demonstrate the relevance of the lipid environment and its resulting physicochemical properties on integrin reconstitution efficiency and its conformational dynamics. We present here an approach to investigate αIIbβ3 in a biomimetic membrane system as a useful platform do dissect disease-relevant integrin mutations and effects on ligand binding in a lipid-specific context, which might be applicable for drug screening.

Expression and functional analysis of a recombinant aquaporin Z from Antarctic Pseudomonas sp. AMS3

“…A PdI value around 0.3 suggests that most of the vesicles in the sample are of a similar size, with some variation in size and/or shape. According to the findings of Janke et al, 31 liposomes and proteoliposomes exhibited greater size uniformity and were well‐suited for analysis using DLS, as evidenced by a lower PdI of approximately 0.24. The distribution may have a relatively narrow peak with a small number of smaller or larger vesicles that contribute to the overall PdI value.…”

Impact of Protein Corona Formation and Polystyrene Nanoparticle Functionalisation on the Interaction with Dynamic Biomimetic Membranes Comprising of Integrin

“…Empty liposomes without any integrin protein were prepared following the same protocol as for the proteoliposomes, adding protein storage buffer instead of purified protein. Validation of successful reconstitution was done as described in previous work and is shown elsewhere [45] …”

“…Materials : Integrin αIIbβ3 was purified as described elsewhere [45] . Bovine serum albumin (BSA) was purchased from Sigma‐Aldrich (Taufkirchen, Germany).…”

“…Reconstitution of integrin αIIbβ3 into liposomes : The reconstitution of integrin αIIbβ3 into liposomes followed a protocol as described by Janke et al [45] . In brief, 1.6 μmol lipids consisting of DOPC, SM, and cholesterol in a ratio of 45 : 30 : 25 mol% were dried under a stream of nitrogen and left in vacuum overnight.…”

“…Crystals were dried under a stream of nitrogen and the surface was exposed for 20 min to UV-ozone. Resonance frequency and dissipation were measured at different harmonics (15,25,35,45,55,65 MHz) simultaneously. Changes in frequency (Δf) and dissipation (ΔD) of the seventh overtone (35 MHz) are presented in the representative QCM graphs.…”