James A. Brozik

The Role of Protein-Protein and Protein-Membrane Interactions on P450 Function.

This symposium summary, sponsored by the ASPET, was held at Experimental Biology 2015 on March 29, 2015, in Boston, Massachusetts. The symposium focused on: 1) the interactions of cytochrome P450s (P450s) with their redox partners; and 2) the role of the lipid membrane in their orientation and stabilization. Two presentations discussed the interactions of P450s with NADPH-P450 reductase (CPR) and cytochrome b5. First, solution nuclear magnetic resonance was used to compare the protein interactions that facilitated either the hydroxylase or lyase activities of CYP17A1. The lyase interaction was stimulated by the presence of b5 and 17α-hydroxypregnenolone, whereas the hydroxylase reaction was predominant in the absence of b5. The role of b5 was also shown in vivo by selective hepatic knockout of b5 from mice expressing CYP3A4 and CYP2D6; the lack of b5 caused a decrease in the clearance of several substrates. The role of the membrane on P450 orientation was examined using computational methods, showing that the proximal region of the P450 molecule faced the aqueous phase. The distal region, containing the substrate-access channel, was associated with the membrane. The interaction of NADPH-P450 reductase (CPR) with the membrane was also described, showing the ability of CPR to “helicopter” above the membrane. Finally, the endoplasmic reticulum (ER) was shown to be heterogeneous, having ordered membrane regions containing cholesterol and more disordered regions. Interestingly, two closely related P450s, CYP1A1 and CYP1A2, resided in different regions of the ER. The structural characteristics of their localization were examined. These studies emphasize the importance of P450 protein organization to their function.

Single particle tracking reveals corralling of a transmembrane protein in a double-cushioned lipid bilayer assembly.

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, 5HT(3A), has been incorporated into several supported membrane bilayer assemblies of increasing complexity. The 5HT(3A) 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 5HT(3A) molecules shows that individual proteins in the double-cushion system have quite high local mobilities but are spatially confined within small corralling domains ( 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.

Coarse-grained model DNA: structure, sequences, stems, circles, hairpins.

A coarse-grained model for DNA that is intended to function realistically at the level of individual bases is reported. The model is composed of residues with up to eight coarse-grained beads each, which is sufficient for DNA-like base stacking and base-base recognition by hydrogen bonding. The beads interact by means of short-ranged pair potentials and a simple implicit solvent model. Movement is simulated by Brownian dynamics without hydrodynamic coupling. The main stabilizing forces are base stacking and hydrogen bonding, as modified by the effects of solvation. Complementary double-stranded chains of such residues form stable double helices over long runs (~10 μs) at or near room temperature, with structural parameters close to those of B-form DNA. Most mismatched chains or mismatched regions within a complementary molecule melt and become disordered. Long-range fluctuations and elastic properties, as measured by bending and twisting persistence lengths, are close to experimental values. Single-stranded chains are flexible, with transient stretches of free bases in equilibrium with globules stabilized by intrastrand stacking and hydrogen bonding. Model DNAs in covalently closed loops form right- or left-handed supercoils, depending on the sign of overtwist or undertwist. Short stem-loop structures melt at elevated temperatures and reanneal when the temperature is carefully lowered. Overall, most qualitative properties of real DNA arise naturally in the model from local interactions at the base-pair level.

Molecular Effects of a Nanocrystalline Quartz Support upon Planar Lipid Bilayers

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 alpha-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.

Synthetic control of intrinsic ground-state defects in a mixed valence quasi-one-dimensional Pt halide chain

Abstract The new linear chain compound [Pt(en)2Cl2][Pt(en)2][Pt(CN)4]2 has been synthesized through two independent methods to yield a quasi-one-dimensional chain with very strong hydrogen bonding interactions. The high charge density wave (CDW) character will be discussed in terms of an unusual hydrogen bonding network which bridges adjacent chains but does not bridge adjacent oxidized and reduced units within a chain. Such a network is unique and breaks from known CDW trends derived from the template effect. The results of the structural analysis by X-ray crystallography have shown that the chains grown from two independent methods are isostructural with one another. Spectroscopic results (resonance Raman and diffuse reflectance) clearly demonstrate the presence of ground-state polaronic defects in crystals grown from solution of stoichiometric amounts of subunits while crystals grown from Pt(IV) rich solutions display a complete absence of ground-state polaronic signatures. These results will be discussed in terms of how one can chemically control the presence and amount of ground-state reduced site defects in the general class of MX materials.

A Guide to Tracking Single Transmembrane Proteins in Supported Lipid Bilayers

The purpose of this chapter is to serve as a guide for those who wish to carry out experiments tracking single transmembrane proteins in planar supported membrane biomimetics. This chapter describes, in detail, the construction of a simple single-molecule microscope, which includes (1) a parts list, (2) an alignment procedure, (3) a calibration procedure, and (4) a procedure for measuring the mechanical stability of the instrument. It also gives procedures for making planar supported POPC bilayers on hydrophilically treated borosilicate and quartz, POPC/PEG-PE cushioned bilayers on hydrophilically treated surfaces, and POPC/PEG-PE cushioned bilayers on BSA passivated substrates. The procedure for the detergent-mediated incorporation of the transmembrane protein 5HT(3A) (a serotonin receptor) is also described and can be used as a starting point for other large non-self-inserting transmembrane proteins. The final experimental section of this chapter details different procedures for data analysis including (1) a quantitative analysis of mean displacements from individually tracked particles, (2) a Gaussian analysis of step-size distributions, (3) the Gaussian analysis of diffusion coefficients from ensembles of transmembrane proteins, and (4) a perspective associated with the interpretation of single-particle tracking data.

Single-Protein Tracking Reveals That NADPH Mediates the Insertion of Cytochrome P450 Reductase into a Biomimetic of the Endoplasmic Reticulum

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.

Substrate Dependent Native Luminescence from Cytochromes P450 3A4, 2C9, and P450cam.

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 (1)ππ* → gs fluorescence and the structured luminescence above 600 nm to a (3)ππ* → gs phosphorescence. These assignments are not associated with the Q-band, and are in violation of Kashas 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.

Conditions for liposome adsorption and bilayer formation on BSA passivated solid supports

Planar 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.

The effect of a phase transition on single molecule tracks of Annexin V in cushioned DMPC assemblies

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.