Patricia M. Dijkman

Microscale thermophoresis quantifies biomolecular interactions under previously challenging conditions

Microscale thermophoresis (MST) allows for quantitative analysis of protein interactions in free solution and with low sample consumption. The technique is based on thermophoresis, the directed motion of molecules in temperature gradients. Thermophoresis is highly sensitive to all types of binding-induced changes of molecular properties, be it in size, charge, hydration shell or conformation. In an all-optical approach, an infrared laser is used for local heating, and molecule mobility in the temperature gradient is analyzed via fluorescence. In standard MST one binding partner is fluorescently labeled. However, MST can also be performed label-free by exploiting intrinsic protein UV-fluorescence. Despite the high molecular weight ratio, the interaction of small molecules and peptides with proteins is readily accessible by MST. Furthermore, MST assays are highly adaptable to fit to the diverse requirements of different biomolecules, such as membrane proteins to be stabilized in solution. The type of buffer and additives can be chosen freely. Measuring is even possible in complex bioliquids like cell lysate allowing close to in vivo conditions without sample purification. Binding modes that are quantifiable via MST include dimerization, cooperativity and competition. Thus, its flexibility in assay design qualifies MST for analysis of biomolecular interactions in complex experimental settings, which we herein demonstrate by addressing typically challenging types of binding events from various fields of life science.

Gating Topology of the Proton-Coupled Oligopeptide Symporters.

Summary Proton-coupled oligopeptide transporters belong to the major facilitator superfamily (MFS) of membrane transporters. Recent crystal structures suggest the MFS fold facilitates transport through rearrangement of their two six-helix bundles around a central ligand binding site; how this is achieved, however, is poorly understood. Using modeling, molecular dynamics, crystallography, functional assays, and site-directed spin labeling combined with double electron-electron resonance (DEER) spectroscopy, we present a detailed study of the transport dynamics of two bacterial oligopeptide transporters, PepTSo and PepTSt. Our results identify several salt bridges that stabilize outward-facing conformations and we show that, for all the current structures of MFS transporters, the first two helices of each of the four inverted-topology repeat units form half of either the periplasmic or cytoplasmic gate and that these function cooperatively in a scissor-like motion to control access to the peptide binding site during transport.

Lipid modulation of early G protein-coupled receptor signalling events.

Upon binding of extracellular ligands, G protein coupled-receptors (GPCRs) initiate signalling cascades by activating heterotrimeric G proteins through direct interactions with the α subunit. While the lipid dependence of ligand binding has previously been studied for one class A GPCR, the neurotensin receptor 1 (NTS1), the role the lipid environment plays in the interaction of activated GPCRs with G proteins is less well understood. It is therefore of interest to understand the balance of lipid interactions required to support both ligand binding and G protein activation, not least since some receptors have multiple locations, and may experience different membrane environments when signalling in the plasma membrane or during endocytosis. Here, using the sensitive biophysical technique of microscale thermophoresis in conjunction with nanodisc lipid bilayer reconstitution, we show that in more native lipid environments rich in phosphatidyl ethanolamine (PE), the Gαi1 subunit has a ~4-fold higher affinity for NTS1 than in the absence of native lipids. The G protein-receptor affinity was further shown to be dependent on the ligand-binding state of the receptor, with potential indication of biased signalling for the known antagonist SR142948A. Gαi1 also showed preferential interaction with empty nanodiscs of native lipid mixtures rich in PE by around 2- to 4-fold over phosphatidyl choline (PC)/phosphatidyl glycerol (PG) lipid mixtures. The lipid environment may therefore play a role in creating favourable micro-environments for efficient GPCR signalling. Our approach combining nanodiscs with microscale thermophoresis will be useful in future studies to elucidate further the complexity of the GPCR interactome.

Reconstitution of Membrane Proteins: A GPCR as an Example

Membrane proteins are the gatekeepers to the cell and are essential to the function of all cells, controlling the flow of molecules and information across the cell membrane. Much effort has been put into the development of systems for studying membrane proteins in simplified environments that nevertheless mimic their native lipid environment. After isolation and production of purified membrane proteins in detergent, it is often necessary to reconstitute them into a lipid structure such as liposome, nanodisc, or lipodisq. Each of these has the advantage of returning the protein to a defined lipid environment, and the choice of system depends on the application. Regardless of the system to be used, the fundamental process involves the removal of detergent and incorporation of the protein into a stable lipid system. This chapter details methodologies we have developed, mainly focussed on the model G protein-coupled receptor (GPCR) neurotensin receptor 1, and the GPCR-homologue and model, bacteriorhopdopsin.

Reconstitution of Membrane Proteins

Membrane proteins are the gatekeepers to the cell and are essential to the function of all cells, controlling the flow of molecules and information across the cell membrane. Much effort has been put into the development of systems for studying membrane proteins in simplified environments that nevertheless mimic their native lipid environment. After isolation and production of purified membrane proteins in detergent, it is often necessary to reconstitute them into a lipid structure such as liposome, nanodisc, or lipodisq. Each of these has the advantage of returning the protein to a defined lipid environment, and the choice of system depends on the application. Regardless of the system to be used, the fundamental process involves the removal of detergent and incorporation of the protein into a stable lipid system. This chapter details methodologies we have developed, mainly focussed on the model G protein-coupled receptor (GPCR) neurotensin receptor 1, and the GPCR-homologue and model, bacteriorhopdopsin.

Lipid-dependent GPCR dimerization

It has been widely demonstrated that G protein-coupled receptors (GPCRs) can form dimers both in vivo and in vitro, a process that has functional consequences. These receptor-receptor interactions take place within a phospholipid bilayer, yet, generally, little is known of the requirements for specific lipids that mediate the dimerization process. Studying this phenomenon in vivo is challenging due to difficulties in modulating the lipid content of cell membranes. Therefore, in this chapter, we describe techniques for reconstitution of GPCRs into model lipid bilayers of defined composition. The concentrations of specific lipids and sterols can be precisely controlled in these liposomes, as well as maintaining an appropriate lipid-protein ratio to avoid artifactual interactions. Receptor dimerization in this system is monitored via Förster resonance energy transfer (FRET), which requires the use of fluorescently labeled receptors. We therefore also include protocols for labeling with appropriate fluorophores and determining the apparent FRET efficiency, a measurement of the extent of receptor dimerization. Understanding the lipid dependence of GPCR dimerization will be key in understanding how this process is regulated in the dynamic heterogeneous environment of the cell membrane.

Interaction of lipids with the neurotensin receptor 1.

Information about lipid-protein interactions for G protein-coupled receptors (GPCRs) is scarce. Here, we use electron spin resonance (ESR) and spin-labelled lipids to study lipid interactions with the rat neurotensin receptor 1 (NTS1). A fusion protein containing rat NTS1 fully able to bind its ligand neurotensin was reconstituted into phosphatidylcholine (PC) bilayers at specific lipid:protein molar ratios. The fraction of motionally restricted lipids in the range of 40:1 to 80:1 lipids per receptor suggested an oligomeric state of the protein, and the result was unaffected by increasing the hydrophobic thickness of the lipid bilayer from C-18 to C-20 or C-22 chain length PC membranes. Comparison of the ESR spectra of different spin-labelled lipids allowed direct measurement of lipid binding constants relative to PC (Kr), with spin-labelled phosphatidylethanolamine (PESL), phosphatidylserine (PSSL), stearic acid (SASL), and a spin labelled cholesterol analogue (CSL) Kr values of 1.05±0.05, 1.92±0.08, 5.20±0.51 and 0.91±0.19, respectively. The results contrast with those from rhodopsin, the only other GPCR studied this way, which has no selectivity for the lipids analysed here. Molecular dynamics simulations of NTS1 in bilayers are in agreement with the ESR data, and point to sites in the receptor where PS could interact with higher affinity. Lipid selectivity could be necessary for regulation of ligand binding, oligomerisation and/or G protein activation processes. Our results provide insight into the potential modulatory mechanisms that lipids can exert on GPCRs.

Dynamic tuneable G protein-coupled receptor monomer-dimer populations

G protein-coupled receptors (GPCRs) are the largest class of membrane receptors, playing a key role in the regulation of processes as varied as neurotransmission and immune response. Evidence for GPCR oligomerisation has been accumulating that challenges the idea that GPCRs function solely as monomeric receptors; however, GPCR oligomerisation remains controversial primarily due to the difficulties in comparing evidence from very different types of structural and dynamic data. Using a combination of single-molecule and ensemble FRET, double electron–electron resonance spectroscopy, and simulations, we show that dimerisation of the GPCR neurotensin receptor 1 is regulated by receptor density and is dynamically tuneable over the physiological range. We propose a “rolling dimer” interface model in which multiple dimer conformations co-exist and interconvert. These findings unite previous seemingly conflicting observations, provide a compelling mechanism for regulating receptor signalling, and act as a guide for future physiological studies.Evidence suggests oligomerisation of G protein-coupled receptors in membranes, but this is controversial. Here, authors use single-molecule and ensemble FRET, and spectroscopy to show that the neurotensin receptor 1 forms multiple dimer conformations that interconvert - “rolling” interfaces.

Single molecule fluorescence for membrane proteins

The cell membrane is a complex milieu of lipids and proteins. In order to understand the behaviour of individual molecules is it often desirable to examine them as purified components in in vitro systems. Here, we detail the creation and use of droplet interface bilayers (DIBs) which, when coupled to TIRF microscopy, can reveal spatiotemporal and kinetic information for individual membrane proteins. A number of steps are required including modification of the protein sequence to enable the incorporation of appropriate fluorescent labels, expression and purification of the membrane protein and subsequent labelling. Following creation of DIBs, proteins are spontaneously incorporated into the membrane where they can be imaged via conventional single molecule TIRF approaches. Using this strategy, in conjunction with step-wise photobleaching, FRET and/or single particle tracking, a host of parameters can be determined such as oligomerisation state and dynamic information. We discuss advantages and limitations of this system and offer guidance for successful implementation of these approaches.