Supported Lipid Bilayers and the Study of Two-Dimensional Binding Kinetics

Abstract

Binding between protein molecules on contacting cells is essential in initiating and regulating several key biological processes. In contrast to interactions between molecules in solution, these events are restricted to the two-dimensional (2D) plane of the meeting cell surfaces. However, converting between the more commonly available binding kinetics measured in solution and the so-called 2D binding kinetics has proven a complicated task since for the latter several factors other than the protein-protein interaction per se have an impact. A few important examples of these are: protein density, membrane fluctuations, force on the bond and the use of auxiliary binding molecules. The development of model membranes, and in particular supported lipid bilayers (SLBs), has made it possible to simplify the studied contact to analyze these effects and to measure 2D binding kinetics of individual protein-protein interactions. We will in this review give an overview of, and discuss, how different SLB systems have been used for this and compare different methods to measure binding kinetics in cell-SLB contacts. Typically, the SLB is functionalized with fluorescently labelled ligands whose interaction with the corresponding receptor on a binding cell can be detected. This interaction can either be studied 1) by an accumulation of ligands in the cell-SLB contact, whose magnitude depends on the density of the proteins and binding affinity of the interaction, or 2) by tracking single ligands in the SLB, which upon interaction with a receptor result in a change of motion of the diffusing ligand. The advantages and disadvantages of other methods measuring 2D binding kinetics will also be discussed and compared to the fluorescence-based methods. Although binding kinetic measurements in cell-SLB contacts have provided novel information on how ligands interact with receptors in vivo the number of these measurements is still limited. This is influenced by the complexity of the system as well as the required experimental time. Moreover, the outcome can vary significantly between studies, highlighting the necessity for continued development of methods to study 2D binding kinetics with higher precision and ease.

Dual-color DNA-PAINT single-particle tracking enables extended studies of membrane protein interactions

“…Proteinprotein interactions in membranes are vital to many aspects of cellular function, including cellular signalling. Hence, experimental techniques that can measure these interactions in situ are highly desirable 18 . We note that in contrast to DNA-PAINT-SPT, most traditional affinity measurement techniques, such as surface plasmon resonance, only give 3D-K D constants and on-rates that cannot easily be converted into their 2D counterparts which are the relevant measures of proteinprotein interactions in membranes 16,[18][19][20] .…”

Phospholipid Epitaxial Assembly Behavior on a Hydrophobic Highly Ordered Pyrolytic Graphite Surface

“…14,15 In the membrane physiological environment provided by supported lipid bilayers (lipids standing on the substrates), the extracellular portion of membrane proteins, peripheral membrane proteins, and lipidanchored proteins have been observed from the top view by AFM or fluorescence microscope, revealing their diffusion property and binding kinetics. 16,17 However, it is hard to directly investigate the transmembrane segment of integral membrane proteins in supported lipid bilayers. Phospholipid assembly of appropriate composition on the substrate with a lying-down phase may help us understand membrane proteins better.…”

“…Plasmalogen is another key component of lipid rafts, participating in the ligand-binding properties of G-protein-coupled receptors. Cholesterol (chol) is also a very important component of biomembranes, accounting for 30% of the total composition, which can adjust the fluidity and stability of biomembranes. , In the membrane physiological environment provided by supported lipid bilayers (lipids standing on the substrates), the extracellular portion of membrane proteins, peripheral membrane proteins, and lipid-anchored proteins have been observed from the top view by AFM or fluorescence microscope, revealing their diffusion property and binding kinetics. , However, it is hard to directly investigate the transmembrane segment of integral membrane proteins in supported lipid bilayers. Phospholipid assembly of appropriate composition on the substrate with a lying-down phase may help us understand membrane proteins better.…”