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Stephen G Sligar

Publications and source records attributed to Stephen G Sligar.

At least 19 recordsLinked to original sources
PubMed2007

Cooperativity in cytochrome P450 3A4: linkages in substrate binding, spin state, uncoupling, and product formation.

Understanding the detailed metabolic mechanisms of membrane-associated cytochromes P450 is often hampered by heterogeneity, ill-defined oligomeric state of the enzyme, and variation in the stoichiometry of the functional P450.reductase complexes in various reconstituted systems. Here, we describe the detailed characterization of a functionally homogeneous 1:1 complex of cytochrome P450 3A4 (CYP3A4) and cytochrome P450 reductase solubilized via self-assembly in a nanoscale phospholipid bilayer. CYP3A4 in this complex showed a nearly complete conversion from the low- to high-spin state when saturated with testosterone (TS) and no noticeable modulation due to the presence of cytochrome P450 reductase. Global analysis of equilibrium substrate binding and steady-state NADPH consumption kinetics provided precise resolution of the fractional contributions to turnover of CYP3A4 intermediates with one, two, or three TS molecules bound. The first binding event accelerates NADPH consumption but does not result in significant product formation due to essentially complete uncoupling. Binding of the second substrate molecule is critically important for catalysis, as the product formation rate reaches a maximum value with two TS molecules bound, whereas the third binding event significantly improves the coupling efficiency of redox equivalent usage with no further increase in product formation rate. The resolution of the fractional contributions of binding intermediates of CYP3A4 into experimentally observed overall spin shift and the rates of steady-state NADPH oxidation and product formation provide new detailed insight into the mechanisms of cooperativity and allosteric regulation in this human cytochrome P450.

Allosteric Regulation↗
PubMed2007

The local phospholipid environment modulates the activation of blood clotting.

Examples abound of membrane-bound enzymes for which the local membrane environment plays an important role, including the ectoenzyme that triggers blood clotting, the plasma serine protease, factor VIIa, bound to the integral membrane protein, tissue factor. The activity of this enzyme complex is markedly influenced by lipid bilayer composition and further by tissue factor partitioning into membrane microdomains on some cell surfaces. Unfortunately, little is known about how membrane microdomain composition controls factor VIIa-tissue factor activity, as reactions catalyzed by membrane-tethered enzymes are typically studied under conditions in which the experimenter cannot control the composition of the membrane in the immediate vicinity of the enzyme. To overcome this problem, we used a nanoscale approach that afforded complete control over the membrane environment surrounding tissue factor by assembling the factor VIIa.tissue factor complex on stable bilayers containing 67 +/- 1 phospholipid molecules/leaflet (Nanodiscs). We investigated how local changes in phospholipid bilayer composition modulate the activity of the factor VIIa.tissue factor complex. We also addressed whether this enzyme requires a pool of membrane-bound protein substrate (factor X) for efficient catalysis, or alternatively if it could efficiently activate factor X, which binds directly to the membrane nanodomain adjacent to tissue factor. We have shown that full proteolytic activity of the factor VIIa.tissue factor complex requires extremely high local concentrations of anionic phospholipids and further that a large pool of membrane-bound factor X is not required to support sustained catalysis.

Blood Coagulation↗
PubMed2006

Resonance surface plasmon spectroscopy: low molecular weight substrate binding to cytochrome p450.

A new detection mechanism has been developed for low molecular weight substrate binding to heme proteins based on resonance localized surface plasmon spectroscopy. Cytochrome P450 has strong electronic transitions in the visible wavelength region. Upon binding of a substrate molecule (e.g., camphor), the absorption band of cytochrome P450 shifts to shorter wavelength. The event of camphor binding to a nanoparticle surface modified with cytochrome P450 protein receptors is monitored using UV-vis spectroscopy. It is observed for the first time that the binding of the substrate molecules to the protein receptor induces a blue-shift in the localized surface plasmon resonance (LSPR) of the nanosensors. The coupling between the molecular resonance of the substrate-free and substrate-bound cytochrome P450 proteins and the nanoparticles' LSPR leads to a highly wavelength-dependent LSPR response. When the LSPR of the nanoparticles is located at a wavelength distant from the cytochrome P450 resonance, an average of approximately 19 nm red-shift is observed upon cytochrome P450 binding to the nanoparticles and a approximately 6 nm blue-shift is observed upon camphor binding However, this response is significantly amplified approximately 3 to 5 times when the LSPR of the nanoparticles is located at a slightly longer wavelength than the cytochrome P450 resonance, that is, a 66.2 nm red-shift upon cytochrome P450 binding and a 34.7 nm blue-shift upon camphor binding. This is the first example of the detection of small molecules binding to a protein modified nanoparticle surface on the basis of LSPR.

Cytochrome P-450 Enzyme System↗
PubMed2006

Structural analysis of nanoscale self-assembled discoidal lipid bilayers by solid-state NMR spectroscopy.

Nanodiscs are an example of discoidal nanoscale self-assembled lipid/protein particles similar to nascent high-density lipoproteins, which reduce the risk of coronary artery disease. The major protein component of high-density lipoproteins is human apolipoprotein A-I, and the corresponding protein component of Nanodiscs is membrane scaffold protein 1 (MSP1), a 200-residue lipid-binding domain of human apolipoprotein A-I. Here we present magic-angle spinning (MAS) solid-state NMR studies of uniformly (13)C,(15)N-labeled MSP1 in polyethylene glycol precipitated Nanodiscs. Two-dimensional MAS (13)C-(13)C correlation spectra show excellent microscopic order of MSP1 in precipitated Nanodiscs. Secondary isotropic chemical shifts throughout the protein are consistent with a predominantly helical structure. Moreover, the backbone conformations of prolines derived from their (13)C chemical shifts are consistent with the molecular belt model but not the picket fence model of lipid-bound MSP1. Overall comparison of experimental spectra and (13)C chemical shifts predicted from several structural models also favors the belt model. Our study thus supports the belt model of Nanodisc structure and demonstrates the utility of MAS NMR to study the structure of high molecular weight lipid-protein complexes.

Computer Simulation↗
PubMed2006

Nanodiscs separate chemoreceptor oligomeric states and reveal their signaling properties.

Bacterial chemoreceptors are transmembrane homodimers that can form trimers, higher order arrays, and extended clusters as part of signaling complexes. Interactions of dimers in oligomers are thought to confer cooperativity and cross-receptor influences as well as a 35-fold gain between ligand binding and altered kinase activity. In addition, higher order interactions among dimers are necessary for the observed patterns of assistance in adaptational modification among different receptors. Elucidating mechanisms underlying these properties will require defining which receptor functions can be performed by dimers and which require specific higher order interactions. However, such an assignment has not been possible. Here, we used Nanodiscs, an emerging technology for manipulating membrane proteins, to prepare small particles of lipid bilayer containing one or only a few chemoreceptor dimers. We found that receptor dimers isolated in individual Nanodiscs were readily modified, bound ligand, and performed transmembrane signaling. However, they were hardly able to activate the chemotaxis histidine kinase. Instead, maximal activation and thus full-range control of kinase occurred preferentially in discs containing approximately three chemoreceptor dimers. The sharp dependence of kinase activation on this number of receptors per dimer implies that the core structural unit of kinase activation and control is a trimer of dimers. Thus, our observations demonstrate that chemoreceptor transmembrane signaling does not require oligomeric organization beyond homodimers and implicate a trimer of dimers as the unit of downstream signaling.

Dimerization↗
PubMed2006

The ferrous-dioxygen intermediate in human cytochrome P450 3A4. Substrate dependence of formation and decay kinetics.

The oxy-ferrous complex is the first of three branching intermediates in the catalytic cycle of cytochrome P450, in which the total efficiency of substrate turnover is curtailed by the side reaction of autoxidation. For human membrane-bound cytochromes P450, the oxy complex is believed to be the primary source of cytotoxic superoxide and peroxide, although information on the properties and stability of this intermediate is lacking. Here we document stopped-flow spectroscopic studies of the formation and decay of the oxy-ferrous complex in the most abundant human cytochrome P450 (CYP3A4) as a function of temperature in the substrate-free and substrate-bound form. CYP3A4 solubilized in purified monomeric form in nanoscale POPC bilayers is functionally and kinetically homogeneous. In substrate-free CYP3A4, the oxy complex is extremely unstable with a half-life of approximately 30 ms at 5 degrees C. Saturation with testosterone or bromocriptine stabilizes the oxy-ferrous intermediate. Comparison of the autoxidation rates with the available data on CYP3A4 turnover kinetics suggests that the oxy complex may be an important route for uncoupling.

Binding Sites↗
PubMed2006

Cytochrome p450 compound I.

Cytochrome P450 enzymes (P450s) comprise a large class of enzymes that effect numerous oxidations in nature. The active oxidants in P450s are thought to be iron(IV)-oxo porphyrin radical cations termed Compounds I, and these intermediates have been sought since the discovery of P450s 40 years ago. We report formation of the Compound I derivative of a P450 enzyme by laser flash photolysis oxidation of the corresponding Compound II species, an iron(IV)-oxo neutral porphyrin intermediate. The Compound II derivative in turn was produced by oxidation of the P450 with peroxynitrite, which effected a net one-electron, oxo-transfer reaction to the iron(III) atom of the resting enzyme. For the P450 studied in this work, CYP119 from the thermophile Sulfolobus solfactaricus, the P450 Compound II derivative was stable for seconds at ambient temperature, and the Compound I transient decayed with a lifetime of ca. 200 ms.

Archaeal Proteins↗
PubMed2006

Assembly of single bacteriorhodopsin trimers in bilayer nanodiscs.

Nanodiscs, phospholipid bilayer assemblies of controlled size, were used to self-assemble bacteriorhodopsin (bR) into single trimers. Self-assembly at optimal bR to Nanodisc and phospholipid stoichiometry yielded particles containing three bR molecules. Analysis of solution small angle X-ray scattering indicated that bacteriorhodopsin is embedded in a discoidal phospholipid bilayer structure. Formation of trimers, as evidenced by visible circular dichroism of the retinal absorbance bands, is facilitated in Nanodiscs at a specific size threshold, suggesting that a critical bilayer area or amount of lipid is necessary to maintain a native oligomeric state. The lipid to bR ratio in the assembly process was also found to be an important factor in determining oligomerization state. These nanoscale bilayers offer the opportunity to understand and control the assembly of oligomeric integral membrane proteins critical to macromolecular recognition and cellular signaling.

Bacteriorhodopsins↗
PubMed2006

The status of high-valent metal oxo complexes in the P450 cytochromes.

The oxidative prowess of the P450 cytochromes in physiological reactions is attributed to the production of a high-valent iron-oxo complex, or Compound I intermediate, in the reaction cycle. Despite many years of study, however, the full electronic description of this fleeting intermediate still remains an active area of study. In this manuscript, the current status of the isolation and characterization of the P450 oxo-Fe(IV) is examined and compared to analogous states in related heme enzymes. In addition, the utilization of cofactor exchange to stabilize high-valent oxo-states in the P450 is addressed. Structural and spectroscopic studies on manganese reconstituted P450, and its corresponding oxo-complex, are presented.

Cytochrome P-450 Enzyme System↗
PubMed2006

Functional reconstitution of Beta2-adrenergic receptors utilizing self-assembling Nanodisc technology.

Integral membrane G protein-coupled receptors (GPCRs) compose the single most prolific class of drug targets, yet significant functional and structural questions remain unanswered for this superfamily. A primary reason for this gap in understanding arises from the difficulty of forming soluble, monodisperse receptor membrane preparations that maintain the transmembrane signaling activity of the receptor and provide robust biophysical and biochemical assay systems. Here we report a technique for self-assembling functional beta2-adrenergic receptor (beta2AR) into a nanoscale phospholipid bilayer system (Nanodisc) that is highly soluble in aqueous solution. The approximately 10-nm nanobilayer particles contain beta2AR in a native-like phospholipid bilayer domain of approximately 100 phospholipid molecules circumferentially bound by a membrane scaffold protein (MSP). The resulting construct allows for access to the physiologically intracellular and extracellular faces of the receptor and thus allows unrestricted access of antagonists, agonists, and G proteins. These Nanodisc-solubilized GPCRs can be directly purified by normal chromatographic procedures. We define the resultant Nanodisc-embedded monomeric beta2AR by antagonist and agonist binding isotherms and demonstrate faithful G protein coupling.

Cell Line↗
PubMed2005

Kinetics of dithionite-dependent reduction of cytochrome P450 3A4: heterogeneity of the enzyme caused by its oligomerization.

To explore the basis of apparent conformational heterogeneity of cytochrome P450 3A4 (CYP3A4), the kinetics of dithionite-dependent reduction was studied in solution, in proteoliposomes, and in Nanodiscs. In CYP3A4 oligomers in solution the kinetics obeys a three-exponential equation with similar amplitudes of each of the phases. Addition of substrate (bromocriptine) displaces the phase distribution toward the slow phase at the expense of the fast one, while the middle phase remains unaffected. The fraction reduced in the fast phase, either with or without substrate, is represented by the low-spin heme protein only, while the slow-reducible fraction is enriched in the high-spin CYP3A4. Upon monomerization by 0.15% Emulgen-913, or by incorporation into Nanodiscs or into large proteoliposomes with a high lipid-to-protein (L/P) ratio (726:1 mol/mol), the kinetics observed in the absence of substrate becomes very rapid and virtually monoexponential. In Nanodiscs and in lipid-rich liposomes bromocriptine decreases the rate of reduction via appearance of the second (slow) phase, the amplitude of which reaches 100% at saturating bromocriptine. In contrast, in P450-rich liposomes (L/P = 112 mol/mol), where the surface molar density of the enzyme is comparable to that observed in liver microsomes, CYP3A4 behaves similarly to that observed in solution. These results suggest that in CYP3A4 oligomers in solution and in the membrane the enzyme is distributed between two persistent conformers with different accessibility of the heme for the reductant (SO*-(2) anion monomer). One of the apparent conformers exists in a substrate-dependent equilibrium between two states with different rate constants of reduction by dithionite, while the second conformer shows no response to substrate binding.

Biopolymers↗
PubMed2005

Thirty years of microbial P450 monooxygenase research: peroxo-heme intermediates--the central bus station in heme oxygenase catalysis.

Oxygen has always been recognized as an essential element of many life forms, initially through its role as a terminal electron acceptor for the energy-generating pathways of oxidative phosphorylation. In 1955, Hayaishi et al. [Mechanism of the pyrocatechase reaction, J. Am. Chem. Soc. 77 (1955) 5450-5451] presented the most important discovery that changed this simplistic view of how Nature uses atmospheric dioxygen. His discovery, the naming and mechanistic understanding of the first "oxygenase" enzyme, has provided a wonderful opportunity and scientific impetus for four decades of researchers. This volume provides an opportunity to recognize the breakthroughs of the "Hayaishi School." Notable have been the prolific contributions of Professor Ishimura et al. [Oxygen and life. Oxygenases, Oxidases and Lipid Mediators, International Congress Series, Elsevier, Amsterdam, 2002], a first-generation Hayaishi product, to characterization of the cytochrome P450 monooxygenases.

Bacterial Proteins↗
PubMed2005

Thermotropic phase transition in soluble nanoscale lipid bilayers.

The role of lipid domain size and protein-lipid interfaces in the thermotropic phase transition of dipalmitoylphosphatidylcholine (DPPC) and dimyristoylphosphatidylcholine (DMPC) bilayers in Nanodiscs was studied using small-angle X-ray scattering (SAXS), differential scanning calorimetry (DSC), and generalized polarization (GP) of the lipophilic probe Laurdan. Nanodiscs are water-soluble, monodisperse, self-assembled lipid bilayers encompassed by a helical membrane scaffold protein (MSP). MSPs of different lengths were used to define the diameter of the Nanodisc lipid bilayer from 76 to 108 A and the number of DPPC molecules from 164 to 335 per discoidal structure. In Nanodiscs of all sizes, the phase transitions were broader and shifted to higher temperatures relative to those observed in vesicle preparations. The size dependences of the transition enthalpies and structural parameters of Nanodiscs reveal the presence of a boundary lipid layer in contact with the scaffold protein encircling the perimeter of the disc. The thickness of this annular layer was estimated to be approximately 15 A, or two lipid molecules. SAXS was used to measure the lateral thermal expansion of Nanodiscs, and a steep decrease of bilayer thickness during the main lipid phase transition was observed. These results provide the basis for the quantitative understanding of cooperative phase transitions in membrane bilayers in confined geometries at the nanoscale.

1,2-Dipalmitoylphosphatidylcholine↗
PubMed2005

The influence of substrate on the spectral properties of oxyferrous wild-type and T252A cytochrome P450-CAM.

To probe whether the nature of the substrate can directly influence the spectral properties of oxyferrous cytochrome P450-CAM, the complex has been investigated in the absence and in the presence of the natural substrate (1R)-camphor (camphor) and of several camphor analogs. The oxyferrous complex of T252A P450-CAM, a mutant lacking the hydroxyl group that forms a hydrogen bond to the heme iron-coordinated dioxygen, has also been studied to gauge the influence of this hydrogen bond. UV-visible absorption and magnetic circular dichroism (MCD) spectra of these oxyferrous adducts prepared and stabilized at -40 degrees C in 60% (v/v) ethylene glycol are generally similar, exhibiting absorption bands at approximately 355, approximately 420, approximately 554, and approximately 585 nm (shoulder) and a characteristic MCD trough at approximately 585 nm. The MCD spectrum of camphor-bound oxyferrous P450-CAM is similar to that of the substrate-free oxyferrous enzyme, but the spectrum of the oxyferrous enzyme differs detectably in the presence of substrate analogs. The spectra of the oxyferrous T252A mutant and wild-type enzyme are overall similar except for Soret band position blue shifts by 2-6 nm for the mutant. 5-Methylenylcamphor (epoxidation substrate) appears to have an anomalous binding mode for the mutant compared with that for the wild-type enzyme. The present results indicate that the structures of the camphor analogs can sensitively influence the physical (spectroscopic) properties of the P450 dioxygen complex and could also affect its reactivity. The ability of substrate to modulate the reactivity of P450 intermediates could be a relevant factor in explaining the remarkable diversity of reactions catalyzed by the enzyme.

Binding Sites↗
PubMed2005

A retinoic acid binding cytochrome P450: CYP120A1 from Synechocystis sp. PCC 6803.

At least 35 cytochrome P450 (P450, CYP) or cytochrome P450-like genes have been identified in 10 cyanobacterial genomes yet none have been functionally characterized. CYP110 and CYP120 represent the two largest cyanobacterial P450 families with 16 and four members, respectively, identified to date. The Synechocystis sp. PCC 6803 CYP120A1 protein sequence shares high degrees of conservation with CYP120A2 from Trichodesmium erythraeum IMS101 and CYP120B1 and CYP120C1 from Nostoc punctiforme PCC 73102. In this communication, we report the cloning, expression, purification, and characterization of CYP120A1 from Synechocystis. Homology modeling predictions of the three-dimensional structure of CYP120A1 coupled with in silico screening for potential substrates and experimental spectroscopic analyses have identified retinoic acid as a compound binding with high affinity to this P450's catalytic site. These characterizations of Synechocystis CYP120A1 lay the initial foundations for understanding the basic role of cytochrome P450s in cyanobacteria and related organisms.

Bacterial Proteins↗
PubMed2004

Molecular dynamics simulations of discoidal bilayers assembled from truncated human lipoproteins.

Human apolipoprotein A-1 (apo A-1) is the major protein component of high-density lipoproteins. The apo A-1 lipid-binding domain was used as a template for the synthesis of amphipathic helical proteins termed membrane scaffold proteins, employed to self-assemble soluble monodisperse discoidal particles called Nanodiscs. In these particles, membrane scaffold proteins surround a lipid bilayer in a belt-like fashion forming bilayer disks of discrete size and composition. Here we investigate the structure of Nanodiscs through molecular dynamics simulations in which Nanodiscs were built from scaffold proteins of various lengths. The simulations showed planar or deformed Nanodiscs depending on optimal length and alignment of the scaffold proteins. Based on mean surface area per lipid calculations, comparison of small-angle x-ray scattering curves, and the relatively planar shape of Nanodiscs made from truncated scaffold proteins, one can conclude that the first 17 to 18 residues of the 200-residue apo A-1 lipid-binding domain are not involved in formation of the protein "belts" surrounding the lipid bilayer. To determine whether the addition of an integral membrane protein has an effect on the overall structure of a Nanodisc, bacteriorhodopsin was embedded into a Nanodisc and simulated using molecular dynamics, revealing a planar disk with a slightly rectangular shape.

1,2-Dipalmitoylphosphatidylcholine↗
PubMed2004

Homotropic cooperativity of monomeric cytochrome P450 3A4 in a nanoscale native bilayer environment.

Mechanistic studies of mammalian cytochrome P450s are often obscured by the phase heterogeneity of solubilized preparations of membrane enzymes. The various protein-protein aggregation states of microsomes, detergent solubilized cytochrome or a family of aqueous multimeric complexes can effect measured substrate binding events as well as subsequent steps in the reaction cycle. In addition, these P450 monooxygenases are normally found in a membrane environment and the bilayer composition and dynamics can also effect these catalytic steps. Here, we describe the structural and functional characterization of a homogeneous monomeric population of cytochrome P450 3A4 (CYP 3A4) in a soluble nanoscale membrane bilayer, or Nanodisc [Nano Lett. 2 (2002) 853]. Cytochrome P450 3A4:Nanodisc assemblies were formed and purified to yield a 1:1 ratio of CYP 3A4 to Nanodisc. Solution small angle X-ray scattering was used to structurally characterize this monomeric CYP 3A4 in the membrane bilayer. The purified CYP 3A4:Nanodiscs showed a heretofore undescribed high level of homotropic cooperativity in the binding of testosterone. Soluble CYP 3A4:Nanodisc retains its known function and shows prototypic hydroxylation of testosterone when driven by hydrogen peroxide. This represents the first functional characterization of a true monomeric preparation of cytochrome P450 monooxygenase in a phospholipid bilayer and elucidates new properties of the monomeric form.

Chromatography, High Pressure Liquid↗