Structural and Interactional Analysis of the Flavonoid Pathway Proteins: Chalcone Synthase, Chalcone Isomerase and Chalcone Isomerase-like Protein.

Chalcone synthase (CHS) and chalcone isomerase (CHI) catalyze the first two committed steps of the flavonoid pathway that plays a pivotal role in the growth and reproduction of land plants, including UV protection, pigmentation, symbiotic nitrogen fixation, and pathogen resistance. Based on the obtained X-ray crystal structures of CHS, CHI, and chalcone isomerase-like protein (CHIL) from the same monocotyledon, Panicum virgatum, along with the results of the steady-state kinetics, spectroscopic/thermodynamic analyses, intermolecular interactions, and their effect on each catalytic step are proposed. In addition, PvCHI's unique activity for both naringenin chalcone and isoliquiritigenin was analyzed, and the observed hierarchical activity for those type-I and -II substrates was explained with the intrinsic characteristics of the enzyme and two substrates. The structure of PvCHS complexed with naringenin supports uncompetitive inhibition. PvCHS displays intrinsic catalytic promiscuity, evident from the formation of p-coumaroyltriacetic acid lactone (CTAL) in addition to naringenin chalcone. In the presence of PvCHIL, conversion of p-coumaroyl-CoA to naringenin through PvCHS and PvCHI displayed ~400-fold increased Vmax with reduced formation of CTAL by 70%. Supporting this model, molecular docking, ITC (Isothermal Titration Calorimetry), and FRET (Fluorescence Resonance Energy Transfer) indicated that both PvCHI and PvCHIL interact with PvCHS in a non-competitive manner, indicating the plausible allosteric effect of naringenin on CHS. Significantly, the presence of naringenin increased the affinity between PvCHS and PvCHIL, whereas naringenin chalcone decreased the affinity, indicating a plausible feedback mechanism to minimize spontaneous incorrect stereoisomers. These are the first findings from a three-body system from the same species, indicating the importance of the macromolecular assembly of CHS-CHI-CHIL in determining the amount and type of flavonoids produced in plant cells.

Thermodynamics and S-Palmitoylation Dependence of Interactions between Human Aquaporin-4 M1 Tetramers in Model Membranes.

Aquaporin-4 (AQP4) is a water channel protein found primarily in the central nervous system (CNS) that helps to regulate water-ion homeostasis. AQP4 exists in two major isoforms: M1 and M23. While both isoforms have a homotetrameric quaternary structure and are functionally identical when transporting water, the M23 isoform forms large protein aggregates known as orthogonal arrays of particles (OAPs). In contrast, the M1 isoform creates a peripheral layer around the outside of these OAPs, suggesting a thermodynamically stable interaction between the two. Structurally, the M1 isoform has an N-terminal tail that is 22 amino acids longer than the M23 isoform and contains two solvent-accessible cysteines available for S-palmitoylation at cysteine-13 (Cys-13) and cysteine-17 (Cys-17) in the amino acid sequence. Earlier work suggests that the palmitoylation of these cysteines might aid in regulating AQP4 assemblies. This work discusses the thermodynamic driving forces for M1 protein-protein interactions and how the palmitoylation state of M1 affects them. Using temperature-dependent single-particle tracking, the standard state free energies, enthalpies, and entropies were measured for these interactions. Furthermore, we present a binding model based on measured thermodynamics and a structural modeling study. The results of this study demonstrate that the M1 isoform will associate with itself according to the following expressions: 2[AQP4-M1]4 ↔ [[AQP4-M1]4]2 when palmitoylated and 3[AQP4-M1]4 ↔ [AQP4-M1]4 + [[AQP4-M1]4]2 ↔ [[AQP4-M1]4]3 when depalmitoylated. This is primarily due to a conformational change induced by adding the palmitic acid groups at Cys-13 and Cys-17 in the N-terminal tails of the homotetramers. In addition, a statistical mechanical model was developed to estimate the Gibbs free energy, enthalpy, and entropy for forming dimers and trimers. These results were in good agreement with experimental values.

Photophysical Rate Constants and Oxygen Dependence for Si and Ge Rhodamine Zwitterions.

The family of group XIV rhodamine zwitterions are fluorescence probes with carbon, silicon, germanium, or tin substituted in the 10-position of the xanthene ring. Because of their inherent near-infrared fluorescence, photostability and high quantum yields in aqueous solutions, the Si and Ge containing fluorophores in this class have become increasingly important for fluorescent labeling of proteins and biological molecules. This study fully characterizes photophysical rates derived from a model consisting of a singlet ground state, the lowest singlet excited state, and the lowest triplet excited state for two exemplar group XIV rhodamine zwitterions, one containing Si and the other Ge. Within a simple Jablonski diagram, all radiative and non-radiative rates, including intersystem crossing and triplet depopulation rates, were measured as a function of oxygen concentration. It was shown that the triplet depopulation rates are intrinsically fast in comparison with traditional xanthene containing fluorophores, probably due to the increased spin-obit coupling from the Si and Ge substitution in the xanthene ring. Dissolved oxygen increases both the intersystem crossing and triplet depopulation rates. Stern-Volmer analysis was conducted to estimate rates of quenching by oxygen. The experimental data was used to estimate the initial rates for reactive oxygen production by Si and Ge containing fluorophores in aqueous solutions containing different concentrations of dissolved O2. These estimates showed a significantly slower initial rate of reactive oxygen production in comparison with rhodamine 6G. This goes a long way to explaining their inherent photostability. Spectroscopic experiments were also conducted in 77 K viscous aqueous glasses where it was observed that the fluorescence spectra remained unchanged, and the quantum yields increased from 0.53 to 0.84 and from 0.52 to 0.89 for the Si and Ge containing fluorophores respectively; no phosphorescence was observed. All intersystem crossing and triplet depopulation rates were measured using fluorescence correlation spectroscopy (FCS) and analyzed using a new method that extrapolated the power dependence of the FCS curves to optical saturation. This method was verified using published data.

Alkynyl Linkers as a Design Tool to Gain Control over the Self-Assembly of Meso-Substituted Porphyrins on HOPG.

Self-assembled monolayers (SAMs) fall generally into two broad categories: those that are covalently bound either to the surface or to each other and those that rely on weaker forces such as hydrogen bonding or van der Waals forces. The engineering of the structure of SAMs formed from weaker forces is an exciting and complex field that often utilizes long alkane substituents bound to core moieties. The core provides the unique optical, electronic, or catalytic property desired, while the interdigitation of the alkane chains provides the means for creating well-regulated patterns of cores on the substrate. This design technique sometimes fails because some of the alkane substituents remain extended into solution rather than become interdigitated on the substrate. One contributor to this is steric hindrance between elements of the core and of the alkane chain. It is shown that the use of an alkyne linker between the core and the alkane chain can, in the case of meso-substituted porphyrins, significantly reduce this steric barrier and allow more stable and predictable surface structures to form. In particular, 5,15-bis(1-octynyl)porphyrin and 5,15-bis(1-tetradecynyl)porphyrin are shown to form significantly more stable SAMs than their alkane-linked counterparts. Scanning tunneling microscopy is used to provide detailed surface structures. Temperature and solution concentration dependence of the surface coverage is also reported. Density functional theory (DFT) is used to determine the energetic effects associated with alkane substitution at both the meso and ß positions and the beneficial energetic effects of the alkyne linker.

A Guide to Tracking Single Membrane Proteins and Their Interactions 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 proteins in planar supported biomimetic membranes. This chapter describes, in detail, the construction of a simple single molecule microscope, which includes: (1) a parts list, (2) temperature control, (3) an alignment procedure, (4) a calibration procedure, and (5) a procedure for measuring the mechanical stability of the instrument. It also gives procedures for making planar supported bilayers on hydrophilically treated borosilicate and quartz. These include (1) POPC bilayers, (2) POPC/PEG-PE cushioned bilayers, (3) POPC/PEG-PE cushioned bilayers on BSA passivated substrates, and (4) a cushioned biomimetic membrane of the endoplasmic reticulum (ER). A procedure for the detergent mediated incorporation of the transmembrane protein 5HT3A (a serotonin receptor) is also described and can be used as a starting point for other large non-self-inserting transmembrane proteins. A procedure for the detergent-free incorporation of cytochrome P450 reductase (CPR) and cytochrome P450 enzymes (P450) into an ER biomimetic is also described. The final experimental section of this chapter details different procedures for data analysis including (1) quantitative analysis of mean squared displacements from individually tracked proteins, (2) gamma distribution analysis of diffusion coefficients from a small ensemble of individually tracked proteins, (3) average mean squared displacement analysis, (4) Gaussian analysis of step-size distributions, (5) Arrhenius analysis of temperature dependent data, (6) the determination of equilibrium constants from a step-size distribution, and (7) a perspective associated with the interpretation of single particle tracking data.

Liquid-cell scanning transmission electron microscopy and fluorescence correlation spectroscopy of DNA-directed gold nanoparticle assemblies.

In the use of solution-based 3D nanoarchitectures for optics, drug delivery, and cancer treatment, the precise nanoparticle architecture morphologies, architecture sizes, interparticle distances, and the assembly stability are all critical to their functionality. 3D nanoparticle architectures in solution are difficult to characterize, as few techniques can provide individualized information on interparticle spacing (defined by linkage molecule), nanoparticle assembly size, morphology, and identification of false aggregation. Bulk characterization techniques, including small angle x-ray scattering, can provide architecture sizes, though they are unable to precisely measure differences within interparticle spacings for individual architectures and can falsely measure assemblies caused by non-linkage grouped nanoparticles. Two solution-based characterization techniques were used to determine which assembly type and linkage length would produce the fastest assembly rate for large DNA-directed gold nanoparticle assemblies. In-situ liquid-cell scanning transmission electron microscopy (LC-STEM), measured interparticle spacings between DNA-functionalized nanoparticles, and fluorescence correlation spectroscopy provided the bulk volume fraction of large and small assemblies for nanoparticle architectures that were assembled using two different types: (1) the hybrid assemblies join two complementary single-stranded DNA linkages, and (2) the bridged assemblies are comprised of single-stranded DNA (bridging component) that is double the length of two different complementary single-stranded DNA-functionalized gold nanoparticles. Assembly times were tested at 24-hrs intervals over 3 days. Statistics derived from the in-situ LC-STEM images provided data for interparticle distance measurements, which identified the fraction of nanoparticles within the images acquired that were at the expected double-stranded DNA-binding distance of the linkages (varied in three distances for each of the two different architectures). In general, longer linkage lengths assembled in the shortest amount of time. The bridged assemblies formed fewer large architectures at 24-hrs but ultimately assembled a greater fraction of nanoparticles, which was due to the longer functionalized DNA lengths for individual nanoparticles. Fluorescence correlation spectroscopy provided a bulk average of the gold nanoparticle assembly sizes over time, which supported the conclusions drawn from the in-situ LC-STEM data. The microscopy provided sub-2 nm precision in the interparticle distances between gold nanoparticles in a solution environment. This coupled microscopy and spectroscopy characterization approach can provide more detailed information than bulk characterization methods.

Dissociation Constants of Cytochrome P450 2C9/Cytochrome P450 Reductase Complexes in a Lipid Bilayer Membrane Depend on NADPH: A Single-Protein Tracking Study.

Cytochrome P450-reductase (CPR) is a versatile NADPH-dependent electron donor located in the cytoplasmic side of the endoplasmic reticulum. It is an electron transferase that is able to deliver electrons to a variety of membrane-bound oxidative partners, including the drug-metabolizing enzymes of the cytochrome P450s (P450). CPR is also stoichiometrically limited compared to its oxidative counterparts, and hypotheses have arisen about possible models that can overcome the stoichiometric imbalance, including quaternary organization of P450 and diffusion-limited models. Described here are results from a single-protein tracking study of fluorescently labeled CPR and cytochrome P450 2C9 (CYP2C9) molecules in which stochastic analysis was used to determine the dissociation constants of CPR/CYP2C9 complexes in a lipid bilayer membrane for the first time. Single-protein trajectories demonstrate the transient nature of these CPR-CYP2C9 interactions, and the measured Kd values are highly dependent on the redox state of CPR. It is shown that CPRox/CYP2C9 complexes have a much higher dissociation constant than CPR2-/CYP2C9 or CPR4-/CYP2C9 complexes, and a model is presented to account for these results. An Arrhenius analysis of diffusion constants was also carried out, demonstrating that the reduced forms of CPR and CYP2C9 interact differently with the biomimetic ER and may, in addition to protein conformational changes, contribute to the observed NADPH-dependent shift in Kd. Finally, it is also shown that the CPRox/CYP2C9 affinity depends on the nature of the ligand, being higher when a substrate is bound, compared to an inhibitor.

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 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.

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.

A fluorescent approach for identifying P2X1 ligands.

There are no commercially available, small, receptor-specific P2X1 ligands. There are several synthetic derivatives of the natural agonist ATP and some structurally-complex antagonists including compounds such as PPADS, NTP-ATP, suramin and its derivatives (e.g. NF279, NF449). NF449 is the most potent and selective ligand, but potencies of many others are not particularly high and they can also act at other P2X, P2Y and non-purinergic receptors. While there is clearly scope for further work on P2X1 receptor pharmacology, screening can be difficult owing to rapid receptor desensitisation. To reduce desensitisation substitutions can be made within the N-terminus of the P2X1 receptor, but these could also affect ligand properties. An alternative is the use of fluorescent voltage-sensitive dyes that respond to membrane potential changes resulting from channel opening. Here we utilised this approach in conjunction with fragment-based drug-discovery. Using a single concentration (300 µM) we identified 46 novel leads from a library of 1443 fragments (hit rate = 3.2%). These hits were independently validated by measuring concentration-dependence with the same voltage-sensitive dye, and by visualising the competition of hits with an Alexa-647-ATP fluorophore using confocal microscopy; confocal yielded kon (1.142 × 10(6) M(-1) s(-1)) and koff (0.136 s(-1)) for Alexa-647-ATP (Kd = 119 nM). The identified hit fragments had promising structural diversity. In summary, the measurement of functional responses using voltage-sensitive dyes was flexible and cost-effective because labelled competitors were not needed, effects were independent of a specific binding site, and both agonist and antagonist actions were probed in a single assay. The method is widely applicable and could be applied to all P2X family members, as well as other voltage-gated and ligand-gated ion channels. This article is part of the Special Issue entitled 'Fluorescent Tools in Neuropharmacology'.

Tracking individual membrane proteins and their biochemistry: The power of direct observation.

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'.

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 (ß-ME) on liposome formation as well as bilayer formation was also explored.

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.

Near native binding of a fluorescent serotonin conjugate to serotonin type 3 receptors.

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 (5HT(3A)). The measured dissociation constant and Hill coefficient are K(d) = 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.

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.

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.

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.

Nanoporous microbead supported bilayers: stability, physical characterization, and incorporation of functional transmembrane proteins.

The introduction of functional transmembrane proteins into supported bilayer-based biomimetic systems presents a significant challenge for biophysics. Among the various methods for producing supported bilayers, liposomal fusion offers a versatile method for the introduction of membrane proteins into supported bilayers on a variety of substrates. In this study, the properties of protein containing unilamellar phosphocholine lipid bilayers on nanoporous silica microspheres are investigated. The effects of the silica substrate, pore structure, and the substrate curvature on the stability of the membrane and the functionality of the membrane protein are determined. Supported bilayers on porous silica microspheres show a significant increase in surface area on surfaces with structures in excess of 10 nm as well as an overall decrease in stability resulting from increasing pore size and curvature. Comparison of the liposomal and detergent-mediated introduction of purified bacteriorhodopsin (bR) and the human type 3 serotonin receptor (5HT3R) are investigated focusing on the resulting protein function, diffusion, orientation, and incorporation efficiency. In both cases, functional proteins are observed; however, the reconstitution efficiency and orientation selectivity are significantly enhanced through detergent-mediated protein reconstitution. The results of these experiments provide a basis for bulk ionic and fluorescent dye-based compartmentalization assays as well as single-molecule optical and single-channel electrochemical interrogation of transmembrane proteins in a biomimetic platform.

Framework model for DNA polymerases.

DNA polymerases are complex machines with both chemical and mechanical functions. Recent crystal structures, ensemble kinetics, and single-molecule investigations have helped to elucidate the main properties of several DNA polymerases, all of which share common structural elements and a common basic mechanism, despite wide variations in amino acid sequence. The framework model is intended to aid in the understanding of these common features (and differences). It defines a class of models that automatically incorporates most of what is known about DNA polymerases within a single theoretical structure so that it is easier to make comparisons between them and to generate detailed models for specific polymerases. The framework model has three main elements: (1) a set of four key variables that describe the important motions within the protein-DNA-nucleotide complex, (2) a complete set of conformational states for the protein-DNA-nucleotide system, and (3) an approximate potential energy surface that controls the motions and transition rates between states. As an example application, we use the general framework ideas to build a detailed model for the HIV reverse transcriptase that is consistent with existing data, and predicts force-velocity curves and stepping-statistics histograms that can be directly compared to experiment.