Cytochrome P450 reductase (CPR) is the redox partner for most human cytochrome P450 enzymes. It is also believed that CPR is an integral membrane protein exclusively. Herein, we report that, contrary to this belief, CPR can exist as a peripheral membrane protein in the absence of NADPH and will transition to an integral membrane protein in the presence of stoichiometric amounts of NADPH or greater. All experiments were performed in a solid-supported cushioned lipid bilayer that closely matched the chemical composition of the human endoplasmic reticulum and served as an ER biomimetic. The phase characteristics and fluidity of the ER biomimetic was characterized with fluorescence micrographs and temperature-dependent fluorescence recovery after photobleaching. The interactions of CPR with the ER biomimetic were directly observed by tracking single CPR molecules using time-lapse single-molecule fluorescence imaging and subsequent analysis of tracks. These studies revealed dramatic changes in diffusion coefficient and the degree of partitioning of CPR as a function of NADPH concentration.

Metalloporphyrin containing proteins, such as cytochrome P450, play a key role in biological systems. The spectroscopic properties of metalloporphyrins have been a subject of intense interest and intense debate for over 50 years. Iron-porphyrins are usually believed to be nonfluorescent. Herein we report that, contrary to this belief, cytochrome P450 heme groups luminesce with enough intensity to be of use in the characterization of these enzymes. To confirm that the emission is from the heme, we destroyed the heme by titration with cumene hydroperoxide and measured the changes in emission upon titration with compounds known to bind to the distal face of the heme in two human cytochrome P450 enzymes, known as CYP3A4 and CYP2C9. The titration curves gave spectral dissociation constants that were not significantly different from those reported using the Soret UV/vis absorbance changes. We have tentatively assigned the broad luminescence at ∼500 nm to a 1ππ* → gs fluorescence and the structured luminescence above 600 nm to a 3ππ* → gs phosphorescence. These assignments are not associated with the Q-band, and are in violation of Kasha’s rule. To illustrate the utility of the emission, we measured spectral dissociation constants for testosterone binding to P450 3A4 in bilayers formed on glass coverslips, a measurement that would be very difficult to make using absorption spectroscopy. Complementary experiments were carried out with water-soluble P450cam.

•Formation of lipid bilayers through the adsorption of SUVs critically depends on the state of the BSA.•Glass substrates coated with a smooth layer of native BSA will readily promote the formation of lipid bilayers.•Glass substrates coated with a smooth layer of heat denatured or chemically reduced BSA will readily promote the adsorption of intact lipid vesicles, but will not promote the formation of lipid bilayers.•Increasing concentrations of B-ME will swell SUVs, except those containing negatively charged lipids, but has no effect on bilayer formation.•Reduced-Heat Denatured BSA has low solubility and will not fully coat a hydrophillically treated glass substrate, but dots the surface with nanometer sized islands, strings, and loopsPlanar solid supported lipid membranes that include an intervening bovine serum albumen (BSA) cushion can greatly reduce undesirable interactions between reconstituted membrane proteins and the underlying substrate. These hetero-self-assemblies reduce frictional coupling by shielding reconstituted membrane proteins from the strong surface charge of the underlying substrate, thereby preventing them from strongly sticking to the substrate themselves. The motivation for this work is to describe the conditions necessary for liposome adsorption and bilayer formation on these hetero-self-assemblies. Described here are experiments that show that the state of BSA is critically important to whether a lipid bilayer is formed or intact liposomes are adsorbed to the BSA passivated surface. It is shown that a smooth layer of native BSA will readily promote lipid bilayer formation while BSA that has been denatured either chemically or by heat will not. Atomic force microscopy (AFM) and fluorescence microscopy was used to characterize the surfaces of native, heat denatured, and chemically reduced BSA. The mobility of several zwitterionic and negatively charged lipid combinations has been measured using fluorescence recovery after photobleaching (FRAP). From these measurements diffusion constants and percent recoveries have been determined and tabulated. The effect of high concentrations of beta-mercaptoethanol (β-ME) on liposome formation as well as bilayer formation was also explored.

Described is the synthesis of 5-hydroxytryptamine-tetramethylrhodamine (5HT∗); an indole nitrogen linked fluorescent conjugate of serotonin. Through a series fluorescence quenching experiments and experiments in the presence of a known competitive antagonist (Granisetron), it was shown that 5HT∗ specifically binds to purified homo-pentameric type-3 human serotonin receptors (5HT3A). The measured dissociation constant and Hill coefficient are Kd = 83 ± 3 nM and n = 3.1 ± 0.3, respectively which is indicative of multi-ligand binding and cooperativity similar to that of unconjugated serotonin.

Using single molecule imaging, this study describes the phase behaviour and mobility of individual transmembrane (TM) proteins and compares those results with the bulk phase behaviour of the biomimetic membrane in which they have been incorporated. To accomplish this a TM protein, Annexin V, was incorporated into a cushioned planar supported 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC)/L-α-lysophosphatidyl-serine (Brain-PS) biomimetic assembly and its mobility between 30 and 16 °C was measured. Fluorescence microscopy and Fluorescence Recovery After Photobleaching (FRAP) were used to verify the structural integrity and the phase behaviour (median melting temperature, TC = 22 °C) of the lipid assembly. The spatial confinement of individual Annexin V molecules was measured in three distinct phase regions: (1) a homogeneous liquid crystalline phase region (Lα) in which Annexin V was unconfined (≥25 °C), (2) a two-phase region (Lα + gel-phase-Pβ′) in which Annexin V displayed intermediate confinement (24–20 °C), and (3) a gel-phase region (Pβ′) with included nanoscopic domains that are enriched with PS and surround a single Annexin V TM protein (19–16 °C); the mobility of Annexin V in these domains is highly confined. At early time lags, Annexin V moves with apparent Brownian-like behaviour at all temperatures but the diffusion coefficients have very different magnitudes and temperature dependence. A possible mechanism for nanoscopic domain formation will be discussed.

A predominate question associated with supported bilayer assemblies containing proteins is whether or not the proteins remain active after incorporation. The major cause for concern is that strong interactions with solid supports can render the protein inactive. To address this question, a large transmembrane protein, the serotonin receptor, 5HT3A, has been incorporated into several supported membrane bilayer assemblies of increasing complexity. The 5HT3A receptor has large extracellular domains on both sides of the membrane, which could cause strong interactions. The bilayer assemblies include a simple POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine) supported planar bilayer, a “single-cushion” POPC bilayer with a PEG (poly(ethylene glycol)) layer between membrane and support, and a “double-cushion” POPC bilayer with both a PEG layer and a layer of BSA (bovine serum albumin). Single-cushion systems are designed to lift the bilayer from the surface, and double-cushion systems are designed to both lift the membrane and passivate the solid support. As in previously reported work, protein mobilities measured by ensemble fluorescence recovery after photobleaching (FRAP) are quite low, especially in the double-cushion system. But single-particle tracking of fluorescent 5HT3A molecules shows that individual proteins in the double-cushion system have quite high local mobilities but are spatially confined within small corralling domains (⟨rC2⟩ ≅ 450 nm). Comparisons with the simple POPC membrane and the single-cushion POPC−PEG membrane reveal that BSA both serves to minimize interactions with the solid support and creates the corrals that reduce the long-range (ensemble averaged) mobility of large transmembrane proteins. These results suggest that in double-cushion assemblies proteins with large extra-membrane domains may remain active and unperturbed despite low bulk diffusion constants.

Supported lipid bilayer membranes play a vital role in a number of applications from biosensors to fundamental studies of membrane proteins. It is widely understood that the underlying solid support in such assemblies causes large perturbations to the lipid bilayer as compared with black lipid membranes, but the exact nature of these effects on the membrane by the solid support is less understood. Here, all-atom molecular dynamics simulations of DLPC, DMPC, POPC, and DEPC on a hydroxylated nanocrystalline α-quartz (011) slab have revealed a pronounced thinning effect. It is shown that this thinning effect proceeds by one of two mechanisms; the first is through a curling of the terminal methyl groups at the interface of opposing leaflets, and the second is through increased interdigitation of the alkyl chains. In all cases, it is shown that the thinning effect is accompanied by a commensurate spreading of the lipid membrane across the quartz substrate. Also, with the introduction of the solid support, a marked asymmetry in a number of structural properties is reported. These asymmetries include (a) the surface areas per lipid, (b) the electron probabilities of the polar headgroups, (c) the radial distributions of the choline groups, and (d) the average orientation of water surrounding the membranes. Finally, asymmetries associated with the different interaction energies within each system studied are reported. These unequal interaction energies lead to a net force holding the membrane to the surface of the support. It was found that direct membrane−substrate interactions play only a minor role in holding the membrane to the surface and it is the interstitial water that dominates these interactions. This is due to the fact that the water throughout the interstitial region displays an average orientational preference that is more favorable (attractive to the membrane and yields a higher number of hydrogen bonds) than water in the external region of the assembly.

•Mini-review with special emphasis on the power of direct observation.•Stochastic fluctuations can be used to build discrete state kinetic models.•Single protein tracking gives a realistic understanding of mass transport.•Identification of individual states can be used to determine chemical potential.•Super-resolution imaging can yield key information about compartmentalization.The advent of single molecule fluorescence microscopy has allowed experimental molecular biophysics and biochemistry to transcend traditional ensemble measurements, where the behavior of individual proteins could not be precisely sampled. The recent explosion in popularity of new super-resolution and super-localization techniques coupled with technical advances in optical designs and fast highly sensitive cameras with single photon sensitivity and millisecond time resolution have made it possible to track key motions, reactions, and interactions of individual proteins with high temporal resolution and spatial resolution well beyond the diffraction limit. Within the purview of membrane proteins and ligand gated ion channels (LGICs), these outstanding advances in single molecule microscopy allow for the direct observation of discrete biochemical states and their fluctuation dynamics. Such observations are fundamentally important for understanding molecular-level mechanisms governing these systems. Examples reviewed here include the effects of allostery on the stoichiometry of ligand binding in the presence of fluorescent ligands; the observation of subdomain partitioning of membrane proteins due to microenvironment effects; and the use of single particle tracking experiments to elucidate characteristics of membrane protein diffusion and the direct measurement of thermodynamic properties, which govern the free energy landscape of protein dimerization. The review of such characteristic topics represents a snapshot of efforts to push the boundaries of fluorescence microscopy of membrane proteins to the absolute limit.This article is part of the Special Issue entitled ‘Fluorescent Tools in Neuropharmacology’.