112 results on '"Sang Choul Im"'
Search Results
2. Kinetic and Structural Characterization of the Effects of Membrane on the Complex of Cytochrome b 5 and Cytochrome c
- Author
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Katherine A. Gentry, Elke Prade, Carlo Barnaba, Meng Zhang, Mukesh Mahajan, Sang-Choul Im, G. M. Anantharamaiah, Satoshi Nagao, Lucy Waskell, and Ayyalusamy Ramamoorthy
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Medicine ,Science - Abstract
Abstract Cytochrome b 5 (cytb 5) is a membrane protein vital for the regulation of cytochrome P450 (cytP450) metabolism and is capable of electron transfer to many redox partners. Here, using cyt c as a surrogate for cytP450, we report the effect of membrane on the interaction between full-length cytb 5 and cyt c for the first time. As shown through stopped-flow kinetic experiments, electron transfer capable cytb 5 - cyt c complexes were formed in the presence of bicelles and nanodiscs. Experimentally measured NMR parameters were used to map the cytb 5-cyt c binding interface. Our experimental results identify differences in the binding epitope of cytb 5 in the presence and absence of membrane. Notably, in the presence of membrane, cytb 5 only engaged cyt c at its lower and upper clefts while the membrane-free cytb 5 also uses a distal region. Using restraints generated from both cytb 5 and cyt c, a complex structure was generated and a potential electron transfer pathway was identified. These results demonstrate the importance of studying protein-protein complex formation in membrane mimetic systems. Our results also demonstrate the successful preparation of novel peptide-based lipid nanodiscs, which are detergent-free and possesses size flexibility, and their use for NMR structural studies of membrane proteins.
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- 2017
- Full Text
- View/download PDF
3. Transmembrane Interactions of Full-length Mammalian Bitopic Cytochrome-P450-Cytochrome-b5 Complex in Lipid Bilayers Revealed by Sensitivity-Enhanced Dynamic Nuclear Polarization Solid-state NMR Spectroscopy
- Author
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Kazutoshi Yamamoto, Marc A. Caporini, Sang-Choul Im, Lucy Waskell, and Ayyalusamy Ramamoorthy
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Medicine ,Science - Abstract
Abstract The dynamic protein-protein and protein-ligand interactions of integral bitopic membrane proteins with a single membrane-spanning helix play a plethora of vital roles in the cellular processes associated with human health and diseases, including signaling and enzymatic catalysis. While an increasing number of high-resolution structural studies of membrane proteins have successfully manifested an in-depth understanding of their biological functions, intact membrane-bound bitopic protein-protein complexes pose tremendous challenges for structural studies by crystallography or solution NMR spectroscopy. Therefore, there is a growing interest in developing approaches to investigate the functional interactions of bitopic membrane proteins embedded in lipid bilayers at atomic-level. Here we demonstrate the feasibility of dynamic nuclear polarization (DNP) magic-angle-spinning NMR techniques, along with a judiciously designed stable isotope labeling scheme, to measure atomistic-resolution transmembrane-transmembrane interactions of full-length mammalian ~72-kDa cytochrome P450-cytochrome b5 complex in lipid bilayers. Additionally, the DNP sensitivity-enhanced two-dimensional 13C/13C chemical shift correlations via proton driven spin diffusion provided distance constraints to characterize protein-lipid interactions and revealed the transmembrane topology of cytochrome b5. The results reported in this study would pave ways for high-resolution structural and topological investigations of membrane-bound full-length bitopic protein complexes under physiological conditions.
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- 2017
- Full Text
- View/download PDF
4. Expression in Escherichia Coli, Purification, and Functional Reconstitution of Human Steroid 5α-Reductases
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Juan Valentín-Goyco, Bing Han, Jie Qiao, Richard J. Auchus, Sang Choul Im, JJ Liu, and Hwei-Ming Peng
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0301 basic medicine ,medicine.medical_specialty ,Protein Folding ,medicine.medical_treatment ,030209 endocrinology & metabolism ,medicine.disease_cause ,Protein Engineering ,Transfection ,Isozyme ,Steroid ,03 medical and health sciences ,0302 clinical medicine ,Endocrinology ,5-alpha Reductase Inhibitors ,3-Oxo-5-alpha-Steroid 4-Dehydrogenase ,Internal medicine ,medicine ,Escherichia coli ,Humans ,Integral membrane protein ,Research Articles ,chemistry.chemical_classification ,Chemistry ,Catabolism ,Recombinant Proteins ,Protein Structure, Tertiary ,Enzyme Activation ,Isoenzymes ,Kinetics ,030104 developmental biology ,Enzyme ,HEK293 Cells ,Biochemistry ,Dihydrotestosterone ,Heterologous expression ,Transformation, Bacterial ,medicine.drug - Abstract
The potent androgen 5α-dihydrotestosterone irreversibly derives from testosterone via the activity of steroid 5α-reductases (5αRs). The major 5αR isoforms in most species, 5αR1 and 5αR2, have not been purified to homogeneity. We report here the heterologous expression of polyhistidine-tagged, codon-optimized human 5αR1 and 5αR2 cDNAs in Escherichia coli. A combination of the nonionic detergents Triton X-100 and Nonidet P-40 enabled solubilization of these extremely hydrophobic integral membrane proteins and facilitated purification with affinity and cation-exchange chromatography methods. For functional reconstitution, we incorporated the purified isoenzymes into Triton X-100-saturated dioleoylphosphatidylcholine liposomes and removed excess detergent with polystyrene beads. Kinetic studies indicated that the 2 isozymes differ in biochemical properties, with 5αR2 having a lower apparent Km for testosterone, androstenedione, progesterone, and 17-hydroxyprogesterone than 5αR1; however, 5αR1 had a greater capacity for steroid conversion, as reflected by a higher Vmax than 5αR2. Both enzymes preferred progesterone as substrate over other steroids, and the catalytic efficiency of purified reconstituted 5αR2 exhibited a sharp pH optimum at pH 5. Intriguingly, we found that the prostate-cancer drug-metabolite 3-keto-∆ 4-abiraterone is metabolized by 5αR1 but not 5αR2, which may serve as a structural basis for isoform selectivity and inhibitor design. The functional characterization results with the purified reconstituted isoenzymes paralleled trends obtained with HEK-293 cell lines stably expressing native 5αR1 and 5αR2. Access to purified human 5αR1 and 5αR2 will advance studies of these important enzymes and might help to clarify their contributions to steroid anabolism and catabolism.
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- 2020
5. Expression, Purification, and Functional Reconstitution of (19)F-labeled Cytochrome b5 in Peptide Nanodiscs for NMR Studies
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Ayyalusamy Ramamoorthy, Jian Wang, Sang Choul Im, Gattadahalli M. Anantharamaiah, Jia Bai, Thirupathi Ravula, and Lucy Waskell
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0301 basic medicine ,Models, Molecular ,Magnetic Resonance Spectroscopy ,Cytochrome ,Size-exclusion chromatography ,Biophysics ,Peptide ,Fluorine-19 NMR ,010402 general chemistry ,01 natural sciences ,Biochemistry ,Article ,03 medical and health sciences ,Cytochrome P-450 Enzyme System ,Protein Domains ,Cytochrome b5 ,Animals ,Lipid bilayer ,Nuclear Magnetic Resonance, Biomolecular ,Nanodisc ,chemistry.chemical_classification ,biology ,Chemistry ,Membrane Proteins ,Cell Biology ,Nuclear magnetic resonance spectroscopy ,0104 chemical sciences ,Transmembrane domain ,030104 developmental biology ,Cytochromes b5 ,Membrane protein ,biology.protein ,Rabbits ,Oxidation-Reduction ,Protein Binding - Abstract
Microsomal cytochrome b5 (cytb5) is a membrane-bound protein capable of donating the second electron to cytochrome P450s (CYP P450s) in the CYP P450s monooxygenase reactions. Recent studies have demonstrated the importance of the transmembrane domain of cytb5 in the interaction with cytP450 by stabilizing its monomeric structure. While recent NMR studies have provided high-resolution insights into the structural interactions between the soluble domains of ~16-kDa cytb5 and ~57-kDa cytP450 in a membrane environment, there is need for studies to probe the residues in the transmembrane region as well as to obtain intermolecular distance constraints to better understand the very large size cytb5-cytP450 complex structure in a near native membrane environment. In this study, we report the expression, purification, functional reconstitution of19F-labeled full-length rabbit cytb5 in peptide based nanodiscs for structural studies using NMR spectroscopy. Size exclusion chromatography, dynamic light scattering, transmission electron microscopy, and NMR experiments show a stable reconstitution of cytb5 in 4F peptide-based lipid-nanodiscs. Our results demonstrate that the use of peptide-nanodiscs containing cytb519F-labeled with 5-fluorotryptophan (5FW) enables, for the first time, the detection of residues from the transmembrane domain in different lipid compositions by solution19F NMR experiments.19F NMR results revealing the interaction of the transmembrane domain of cytb5 with the full-length rabbit Cytochrome P450 2B4 (CYP2B4) are also presented. We expect the results presented in this study to be useful to devise approaches to probe the structure, dynamics and functional roles of transmembrane domains of a membrane protein, and also to measure intermolecular19F-19F distance constraints to determine the structural interactions between the transmembrane domains.
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- 2020
6. A Minimal Functional Complex of Cytochrome P450 and FBD of Cytochrome P450 Reductase in Nanodiscs
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Mukesh Mahajan, Katherine A. Gentry, Sang Choul Im, Ayyalusamy Ramamoorthy, Gattadahalli M. Anantharamaiah, Lucy Waskell, Meng Zhang, and Elke Prade
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Models, Molecular ,0301 basic medicine ,Flavin Mononucleotide ,010402 general chemistry ,Redox ,01 natural sciences ,Article ,Catalysis ,Electron transfer ,03 medical and health sciences ,Cytochrome P-450 Enzyme System ,Lipid bilayer ,biology ,Mechanism (biology) ,Chemistry ,Cytochrome P450 reductase ,Cytochrome P450 ,General Chemistry ,Nuclear magnetic resonance spectroscopy ,General Medicine ,Nanostructures ,0104 chemical sciences ,030104 developmental biology ,Membrane protein ,biology.protein ,Biophysics ,Peptides ,Oxidation-Reduction - Abstract
Structural interactions that enable electron transfer to cytochrome-P450 (CYP450) from its redox partner CYP450-reductase (CPR) are a vital prerequisite for its catalytic mechanism. The first structural model for the membrane-bound functional complex to reveal interactions between the full-length CYP450 and a minimal domain of CPR is now reported. The results suggest that anchorage of the proteins in a lipid bilayer is a minimal requirement for CYP450 catalytic function. Akin to cytochrome-b5 (cyt-b5 ), Arg 125 on the C-helix of CYP450s is found to be important for effective electron transfer, thus supporting the competitive behavior of redox partners for CYP450s. A general approach is presented to study protein-protein interactions combining the use of nanodiscs with NMR spectroscopy and SAXS. Linking structural details to the mechanism will help unravel the xenobiotic metabolism of diverse microsomal CYP450s in their native environment and facilitate the design of new drug entities.
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- 2018
7. Cytochrome-P450-Induced Ordering of Microsomal Membranes Modulates Affinity for Drugs
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Lucy Waskell, Bikash R. Sahoo, Ayyalusamy Ramamoorthy, Sang Choul Im, Carlo Barnaba, Gattadahalli M. Anantharamaiah, Thirupathi Ravula, and Ilce G. Medina-Meza
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0301 basic medicine ,Cytochrome ,Molecular Dynamics Simulation ,Endoplasmic Reticulum ,010402 general chemistry ,01 natural sciences ,Catalysis ,Article ,Membrane Lipids ,03 medical and health sciences ,Cytochrome P-450 Enzyme System ,Animals ,Humans ,Lipid bilayer ,biology ,Chemistry ,Endoplasmic reticulum ,Cytochrome P450 ,Membrane Proteins ,General Chemistry ,Raft ,General Medicine ,Ligand (biochemistry) ,Sphingomyelins ,0104 chemical sciences ,Kinetics ,Membrane ,030104 developmental biology ,biology.protein ,Biophysics ,Nanoparticles ,lipids (amino acids, peptides, and proteins) ,Sphingomyelin ,Protein Binding - Abstract
Although membrane environment is known to boost drug metabolism by mammalian cytochrome P450s, the factors that stabilize the structural folding and enhance protein function are unclear. In this study, we use peptide-based lipid nanodiscs to "trap" the lipid boundaries of microsomal cytochrome P450 2B4. We report the first evidence that CYP2B4 is able to induce the formation of raft domains in a biomimetic compound of the endoplasmic reticulum. NMR experiments were used to identify and quantitatively determine the lipids present in nanodiscs. A combination of biophysical experiments and molecular dynamics simulations revealed a sphingomyelin binding region in CYP2B4. The protein-induced lipid raft formation increased the thermal stability of P450 and dramatically altered ligand binding kinetics of the hydrophilic ligand BHT. These results unveil membrane/protein dynamics that contribute to the delicate mechanism of redox catalysis in lipid membrane.
- Published
- 2018
8. Substrate mediated redox partner selectivity of cytochrome P450
- Author
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Ayyalusamy Ramamoorthy, Meng Zhang, Katherine A. Gentry, Lucy Waskell, and Sang Choul Im
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0301 basic medicine ,Gating ,Plasma protein binding ,Ligands ,Redox ,Article ,Catalysis ,Substrate Specificity ,03 medical and health sciences ,Protein Domains ,Cyclohexanes ,Materials Chemistry ,Animals ,Cytochrome P450 Family 2 ,Nuclear Magnetic Resonance, Biomolecular ,Ternary complex ,NADPH-Ferrihemoprotein Reductase ,biology ,Chemistry ,Metals and Alloys ,Benzphetamine ,Cytochrome P450 ,Substrate (chemistry) ,General Chemistry ,Butylated Hydroxytoluene ,Rats ,Surfaces, Coatings and Films ,Electronic, Optical and Magnetic Materials ,Cytochromes b5 ,Methoxyflurane ,030104 developmental biology ,Models, Chemical ,Multiprotein Complexes ,Ceramics and Composites ,biology.protein ,Biophysics ,Aryl Hydrocarbon Hydroxylases ,Rabbits ,Protein Multimerization ,Selectivity ,Function (biology) ,Protein Binding - Abstract
Investigating the interplay between cytochrome-P450 and its redox partners (CPR and cytochrome-b(5)) is vital for understanding the metabolism of most hydrophobic drugs. Dynamic structural interactions with the ternary complex, with and without substrates, captured by NMR reveal a gating mechanism for redox partners to promote P450 function.
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- 2018
9. Lipid-exchange in nanodiscs discloses membrane boundaries of cytochrome-P450 reductase
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Ayyalusamy Ramamoorthy, Thirupathi Ravula, Carlo Barnaba, Lucy Waskell, Sang Choul Im, Ilce G. Medina-Meza, and Gattadahalli M. Anantharamaiah
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0301 basic medicine ,Oxygenase ,Flavin Mononucleotide ,Protein Conformation ,Article ,Fluorescence ,Catalysis ,03 medical and health sciences ,chemistry.chemical_compound ,Ethanolamine ,Protein structure ,Materials Chemistry ,Animals ,Transferase ,NADPH-Ferrihemoprotein Reductase ,Phosphatidylethanolamines ,Metals and Alloys ,Membrane Proteins ,Cytochrome P450 reductase ,Membranes, Artificial ,General Chemistry ,Nanostructures ,Surfaces, Coatings and Films ,Electronic, Optical and Magnetic Materials ,030104 developmental biology ,Membrane ,chemistry ,Membrane protein ,Flavin-Adenine Dinucleotide ,Ceramics and Composites ,Microsome ,Biophysics ,Cattle ,Peptides - Abstract
Lipids are critical for the function of membrane proteins. NADPH-cytochrome-P450-reductase, the sole electron transferase for microsomal oxygenases, possesses a conformational dynamics entwined with its topology. Here, we use peptide-nanodiscs to unveil cytochrome-P450-reductase's lipid boundaries, demonstrating a protein-driven enrichment of ethanolamine lipids (by 25%) which ameliorates by 3-fold CPR's electron-transfer ability.
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- 2018
10. The FMN '140s Loop' of Cytochrome P450 Reductase Controls Electron Transfer to Cytochrome P450
- Author
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Sang Choul Im, Lucy Waskell, and Freeborn Rwere
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Cytochrome ,Semiquinone ,anionic semiquinone ,Flavin Mononucleotide ,Protein Conformation ,Mutant ,P450 BM3 ,redox potential ,cytochrome b5 ,Biology (General) ,Spectroscopy ,cytochrome P450 reductase ,biology ,Chemistry ,Communication ,Cytochrome P450 reductase ,General Medicine ,Computer Science Applications ,hydroquinone ,Aryl Hydrocarbon Hydroxylases ,Rabbits ,Oxidation-Reduction ,Cytochrome-B(5) Reductase ,Protein Binding ,QH301-705.5 ,cytochrome P450 ,Glycine ,Electrons ,Catalysis ,Electron Transport ,Inorganic Chemistry ,FMN binding ,Microsomes ,Cytochrome b5 ,Animals ,Amino Acid Sequence ,Physical and Theoretical Chemistry ,Cytochrome P450 Family 2 ,QD1-999 ,Molecular Biology ,NADPH-Ferrihemoprotein Reductase ,Organic Chemistry ,Wild type ,Cytochrome P450 ,Kinetics ,Cytochromes b5 ,Mutagenesis, Site-Directed ,biology.protein ,Biophysics ,Mutant Proteins - Abstract
Cytochrome P450 reductase (CYPOR) provides electrons to all human microsomal cytochrome P450s (cyt P450s). The length and sequence of the “140s” FMN binding loop of CYPOR has been shown to be a key determinant of its redox potential and activity with cyt P450s. Shortening the “140s loop” by deleting glycine-141(ΔGly141) and by engineering a second mutant that mimics flavo-cytochrome P450 BM3 (ΔGly141/Glu142Asn) resulted in mutants that formed an unstable anionic semiquinone. In an attempt to understand the molecular basis of the inability of these mutants to support activity with cyt P450, we expressed, purified, and determined their ability to reduce ferric P450. Our results showed that the ΔGly141 mutant with a very mobile loop only reduced ~7% of cyt P450 with a rate similar to that of the wild type. On the other hand, the more stable loop in the ΔGly141/Glu142Asn mutant allowed for ~55% of the cyt P450 to be reduced ~60% faster than the wild type. Our results reveal that the poor activity of the ΔGly141 mutant is primarily accounted for by its markedly diminished ability to reduce ferric cyt P450. In contrast, the poor activity of the ΔGly141/Glu142Asn mutant is presumably a consequence of the altered structure and mobility of the “140s loop”.
- Published
- 2021
11. Transmembrane Interactions of Full-length Mammalian Bitopic Cytochrome-P450-Cytochrome-b5 Complex in Lipid Bilayers Revealed by Sensitivity-Enhanced Dynamic Nuclear Polarization Solid-state NMR Spectroscopy
- Author
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Ayyalusamy Ramamoorthy, Lucy Waskell, Kazutoshi Yamamoto, Sang Choul Im, and Marc A. Caporini
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0301 basic medicine ,Models, Molecular ,Cytochrome ,Protein Conformation ,Science ,Lipid Bilayers ,010402 general chemistry ,01 natural sciences ,Article ,03 medical and health sciences ,Protein structure ,Cytochrome P-450 Enzyme System ,Cytochrome b5 ,Animals ,Humans ,Amino Acid Sequence ,Carbon-13 Magnetic Resonance Spectroscopy ,Lipid bilayer ,Nuclear Magnetic Resonance, Biomolecular ,Multidisciplinary ,biology ,Chemistry ,Nuclear magnetic resonance spectroscopy ,Transmembrane protein ,0104 chemical sciences ,030104 developmental biology ,Cytochromes b5 ,Membrane protein ,Biochemistry ,Membrane topology ,Multiprotein Complexes ,biology.protein ,Biophysics ,Medicine ,Protein Binding - Abstract
The dynamic protein-protein and protein-ligand interactions of integral bitopic membrane proteins with a single membrane-spanning helix play a plethora of vital roles in the cellular processes associated with human health and diseases, including signaling and enzymatic catalysis. While an increasing number of high-resolution structural studies of membrane proteins have successfully manifested an in-depth understanding of their biological functions, intact membrane-bound bitopic protein-protein complexes pose tremendous challenges for structural studies by crystallography or solution NMR spectroscopy. Therefore, there is a growing interest in developing approaches to investigate the functional interactions of bitopic membrane proteins embedded in lipid bilayers at atomic-level. Here we demonstrate the feasibility of dynamic nuclear polarization (DNP) magic-angle-spinning NMR techniques, along with a judiciously designed stable isotope labeling scheme, to measure atomistic-resolution transmembrane-transmembrane interactions of full-length mammalian ~72-kDa cytochrome P450-cytochrome b5 complex in lipid bilayers. Additionally, the DNP sensitivity-enhanced two-dimensional 13C/13C chemical shift correlations via proton driven spin diffusion provided distance constraints to characterize protein-lipid interactions and revealed the transmembrane topology of cytochrome b5. The results reported in this study would pave ways for high-resolution structural and topological investigations of membrane-bound full-length bitopic protein complexes under physiological conditions.
- Published
- 2017
12. Protonation of the Hydroperoxo Intermediate of Cytochrome P450 2B4 Is Slower in the Presence of Cytochrome P450 Reductase Than in the Presence of Cytochrome b5
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R. David Britt, Sang Choul Im, Ryan C. Kunz, Lucy Waskell, Jarett Wilcoxen, Joseph E. Darty, Stephen W. Ragsdale, and Naw May Pearl
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0301 basic medicine ,Electrons ,Protonation ,Reductase ,Photochemistry ,Models, Biological ,Biochemistry ,Redox ,Substrate Specificity ,03 medical and health sciences ,chemistry.chemical_compound ,Cytochrome b5 ,medicine ,Animals ,Cytochrome P450 Family 2 ,Heme ,NADPH-Ferrihemoprotein Reductase ,030102 biochemistry & molecular biology ,biology ,Electron Spin Resonance Spectroscopy ,Cytochrome P450 reductase ,Cytochrome P450 ,NAD ,Kinetics ,Cytochromes b5 ,chemistry ,Biocatalysis ,biology.protein ,Ferric ,Aryl Hydrocarbon Hydroxylases ,Hydrogenation ,Rabbits ,Protons ,Oxidation-Reduction ,Protein Binding ,medicine.drug - Abstract
Microsomal cytochromes P450 (P450) require two electrons and two protons for the oxidation of substrates. Although the two electrons can be provided by cytochrome P450 reductase, the second electron can also be donated by cytochrome b5 (b5). The steady-state activity of P450 2B4 is increased up to 10-fold by b5. To improve our understanding of the molecular basis of the stimulatory effect of b5 and to test the hypothesis that b5 stimulates catalysis by more rapid protonation of the anionic ferric hydroperoxo heme intermediate of P450 (Fe3+OOH)− and subsequent formation of the active oxidizing species (Fe+4═O POR•+), we have freeze-quenched the reaction mixture during a single turnover following reduction of oxyferrous P450 2B4 by each of its redox partners, b5 and P450 reductase. The electron paramagnetic resonance spectra of the freeze-quenched reaction mixtures lacked evidence of a hydroperoxo intermediate when b5 was the reductant presumably because hydroperoxo protonation and catalysis occurred within...
- Published
- 2016
13. Role of the Proximal Cysteine Hydrogen Bonding Interaction in Cytochrome P450 2B4 Studied by Cryoreduction, Electron Paramagnetic Resonance, and Electron–Nuclear Double Resonance Spectroscopy
- Author
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Lucy Waskell, William A. Gunderson, Muralidharan Shanmugam, Roman Davydov, Sang Choul Im, Brian M. Hoffman, and Naw May Pearl
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0301 basic medicine ,Crystallography, X-Ray ,010402 general chemistry ,Photochemistry ,01 natural sciences ,Biochemistry ,Article ,law.invention ,03 medical and health sciences ,law ,Cysteine ,Cytochrome P450 Family 2 ,Electron paramagnetic resonance ,Spectroscopy ,Hyperfine structure ,Cryopreservation ,030102 biochemistry & molecular biology ,biology ,Chemistry ,Hydrogen bond ,Electron Spin Resonance Spectroscopy ,Cytochrome P450 ,Hydrogen Bonding ,0104 chemical sciences ,Catalytic cycle ,Radiolysis ,biology.protein ,Aryl Hydrocarbon Hydroxylases - Abstract
Crystallographic studies have shown that the F429H mutation of cytochrome P450 2B4 introduces an H-bond between His429 and the proximal thiolate ligand, Cys436, without altering the protein fold but sharply decreases the enzymatic activity and stabilizes the oxyferrous P450 2B4 complex. To characterize the influence of this hydrogen bond on the states of the catalytic cycle, we have used radiolytic cryoreduction combined with electron paramagnetic resonance (EPR) and (electron-nuclear double resonance (ENDOR) spectroscopy to study and compare their characteristics for wild-type (WT) P450 2B4 and the F429H mutant. (i) The addition of an H-bond to the axial Cys436 thiolate significantly changes the EPR signals of both low-spin and high-spin heme-iron(III) and the hyperfine couplings of the heme-pyrrole (14)N but has relatively little effect on the (1)H ENDOR spectra of the water ligand in the six-coordinate low-spin ferriheme state. These changes indicate that the H-bond introduced between His and the proximal cysteine decreases the extent of S → Fe electron donation and weakens the Fe(III)-S bond. (ii) The added H-bond changes the primary product of cryoreduction of the Fe(II) enzyme, which is trapped in the conformation of the parent Fe(II) state. In the wild-type enzyme, the added electron localizes on the porphyrin, generating an S = (3)/2 state with the anion radical exchange-coupled to the Fe(II). In the mutant, it localizes on the iron, generating an S = (1)/2 Fe(I) state. (iii) The additional H-bond has little effect on g values and (1)H-(14)N hyperfine couplings of the cryogenerated, ferric hydroperoxo intermediate but noticeably slows its decay during cryoannealing. (iv) In both the WT and the mutant enzyme, this decay shows a significant solvent kinetic isotope effect, indicating that the decay reflects a proton-assisted conversion to Compound I (Cpd I). (v) We confirm that Cpd I formed during the annealing of the cryogenerated hydroperoxy intermediate and that it is the active hydroxylating species in both WT P450 2B4 and the F429H mutant. (vi) Our data also indicate that the added H-bond of the mutation diminishes the reactivity of Cpd I.
- Published
- 2016
14. Effect of polymer charge on functional reconstitution of membrane proteins in polymer nanodiscs
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Ayyalusamy Ramamoorthy, Thirupathi Ravula, Nathaniel Z. Hardin, Sang Choul Im, Lucy Waskell, and Jia Bai
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0301 basic medicine ,chemistry.chemical_classification ,Metals and Alloys ,Charge (physics) ,General Chemistry ,Polymer ,010402 general chemistry ,01 natural sciences ,Catalysis ,Article ,0104 chemical sciences ,Surfaces, Coatings and Films ,Electronic, Optical and Magnetic Materials ,03 medical and health sciences ,030104 developmental biology ,chemistry ,Membrane protein ,Materials Chemistry ,Ceramics and Composites ,Biophysics - Abstract
Although there is a growing interest in using polymer lipid-nanodiscs, the polymer charge poses limitations for studies on membrane proteins. Here, we demonstrate the functional reconstitution of a large soluble-domain containing positively-charged ∼57 kDa cytochrome-P450 and negatively-charged ∼16 kDa cytochrome-b5 in lipid-nanodiscs, and the role of the polymer charge for high-resolution studies on membrane proteins.
- Published
- 2018
15. Structural and kinetic studies of Asp632 mutants and fully-reduced NADPH-cytochrome P450 oxidoreductase define the role of Asp632 loop dynamics in control of NADPH binding and hydride transfer
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Jung-Ja P. Kim, Anna Shen, Chuanwu Xia, Sang Choul Im, Freeborn Rwere, and Lucy Waskell
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0301 basic medicine ,Models, Molecular ,Stereochemistry ,medicine.drug_class ,Flavin Mononucleotide ,Protein Conformation ,Carboxamide ,Flavin group ,Crystallography, X-Ray ,Biochemistry ,Article ,Electron Transport ,03 medical and health sciences ,Electron transfer ,Protein structure ,Oxidoreductase ,medicine ,Animals ,Point Mutation ,NADPH-Ferrihemoprotein Reductase ,chemistry.chemical_classification ,Chemistry ,Electron acceptor ,Electron transport chain ,Adenosine Monophosphate ,Rats ,Kinetics ,030104 developmental biology ,Amino Acid Substitution ,Flavin-Adenine Dinucleotide ,NADPH binding ,Oxidation-Reduction ,NADP ,Protein Binding - Abstract
Conformational changes of NADPH-cytochrome P450 oxidoreductase (CYPOR) associated with electron transfer from NADPH to electron acceptors via FAD and FMN have been investigated through structural studies of the 4-electron-reduced NADP+-bound enzyme and kinetic and structural studies of mutants affecting the conformation of the mobile Gly631-Asn635 loop (Asp632 loop). The structure of 4-electron-reduced, NADP+-bound wild type CYPOR shows the plane of the nicotinamide ring positioned perpendicular to the FAD isoalloxazine with its carboxamide group forming H-bonds with N1 of the flavin ring and the Thr535 hydroxyl group. In the reduced enzyme, the C8-C8 atoms of the two flavin rings are ~1 Å closer compared to the fully oxidized and 1-electron-reduced structures, which suggests that flavin reduction facilitates interflavin electron transfer. Structural and kinetic studies of mutants, Asp632Ala, Asp632Phe, Asp632Asn, and Asp632Glu demonstrate that the carboxyl group of Asp632 is important for stabilizing the Asp632 loop in a retracted position that is required for the binding of the NADPH ribityl-nicotinamide in a hydride-transfer-competent conformation. Structures of the mutants and reduced wild type CYPOR permit us to identify a possible pathway for NADP(H) binding/release to/from CYPOR. Asp632 mutants unable to form stable H-bonds with the backbone amides of Arg634, Asn635, and Met636, exhibit decreased catalytic activity and severely impaired hydride transfer from NADPH to FAD, but leave interflavin electron transfer intact. Intriguingly, the Arg634Ala mutation slightly increases the cytochrome P450 2B4 activity. We propose that Asp632 loop movement, in addition to facilitating NADP(H) binding and release, participates in domain movements modulating interflavin electron transfer.
- Published
- 2018
16. Structure of cytochrome P450 2B4 with an acetate ligand and an active site hydrogen bond network similar to oxyferrous P450cam
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Jeanne A. Stuckey, Weishu Bu, Jennifer L. Meagher, Yuting Yang, Sang Choul Im, and Lucy Waskell
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0301 basic medicine ,Stereochemistry ,Protein Conformation ,Static Electricity ,chemistry.chemical_element ,Glutamic Acid ,Acetates ,Crystallography, X-Ray ,Ligands ,Biochemistry ,Oxygen ,Article ,Inorganic Chemistry ,03 medical and health sciences ,Protein structure ,Catalytic Domain ,Animals ,Ferrous Compounds ,Threonine ,Cytochrome P450 Family 2 ,Conformational isomerism ,030102 biochemistry & molecular biology ,biology ,Hydrogen bond ,Ligand ,Active site ,Hydrogen Bonding ,Bond length ,chemistry ,biology.protein ,Aryl Hydrocarbon Hydroxylases ,Rabbits - Abstract
Superposition of the active site of acetate-bound P4502B4 and oxyferrous P450cam. Bond lengths between the heme iron, the sixth ligand, and the hydroxyl of the conserved threonine are shown. Note that different threonine rotamers form the hydrogen bonds to the acetate and oxygen. [Image: see text]
- Published
- 2017
17. Membrane Environment Drives Cytochrome P450’s Spin Transition and its Interaction with Cytochrome b5
- Author
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Gattadahalli M. Anantharamaiah, Ayyalusamy Ramamoorthy, Lucy Waskell, Carlo Barnaba, Mukesh Mahajan, Thirupathi Ravula, and Sang Choul Im
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0301 basic medicine ,Models, Molecular ,Stereochemistry ,Spin transition ,010402 general chemistry ,01 natural sciences ,Catalysis ,Article ,03 medical and health sciences ,chemistry.chemical_compound ,Cytochrome C1 ,Cytochrome P-450 Enzyme System ,Cytochrome b5 ,Materials Chemistry ,medicine ,Lipid bilayer ,Heme ,biology ,Chemistry ,Metals and Alloys ,Cytochrome P450 ,General Chemistry ,0104 chemical sciences ,Surfaces, Coatings and Films ,Electronic, Optical and Magnetic Materials ,030104 developmental biology ,Membrane ,Cytochromes b5 ,Ceramics and Composites ,biology.protein ,Ferric ,medicine.drug - Abstract
Heme's spin-multiplicity is key in determining the enzymatic function of cytochrome P450 (cytP450). The origin of the low-spin state in ferric P450 is still under debate. Here, we report the first experimental demonstration of P450's membrane interaction altering its spin equilibrium which is accompanied by a stronger affinity for cytochrome b5. These results highlight the importance of lipid membrane for the function of P450.
- Published
- 2017
18. Structural and Functional Characterization of a Cytochrome P450 2B4 F429H Mutant with an Axial Thiolate–Histidine Hydrogen Bond
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Weishu Bu, Jennifer L. Meagher, Dandamudi Usharani, Naw May Pearl, Michael Tarasev, Yuting Yang, Jeanne A. Stuckey, Cuthbert Sun, Haoming Zhang, Sason Shaik, Freeborn Rwere, Sang Choul Im, and Lucy Waskell
- Subjects
Models, Molecular ,Heme binding ,Stereochemistry ,Protein Conformation ,Phenylalanine ,Resonance Raman spectroscopy ,Heme ,Crystallography, X-Ray ,Biochemistry ,Article ,Substrate Specificity ,Electron Transport ,chemistry.chemical_compound ,Protein structure ,Histidine ,Cytochrome P450 Family 2 ,NADPH-Ferrihemoprotein Reductase ,Binding Sites ,biology ,Hydrogen bond ,Cytochrome P450 ,Hydrogen Bonding ,Electron transport chain ,Recombinant Proteins ,Kinetics ,Cytochromes b5 ,chemistry ,Amino Acid Substitution ,Structural Homology, Protein ,biology.protein ,Mutagenesis, Site-Directed ,Thermodynamics ,Mutant Proteins ,Aryl Hydrocarbon Hydroxylases ,Oxidation-Reduction - Abstract
The structural basis of the regulation of microsomal cytochrome P450 (P450) activity was investigated by mutating the highly conserved heme binding motif residue, Phe429, on the proximal side of cytochrome P450 2B4 to a histidine. Spectroscopic, pre-steady-state and steady-state kinetic, thermodynamic, theoretical, and structural studies of the mutant demonstrate that formation of an H-bond between His429 and the unbonded electron pair of the Cys436 axial thiolate significantly alters the properties of the enzyme. The mutant lost >90% of its activity; its redox potential was increased by 87 mV, and the half-life of the oxyferrous mutant was increased ∼37-fold. Single-crystal electronic absorption and resonance Raman spectroscopy demonstrated that the mutant was reduced by a small dose of X-ray photons. The structure revealed that the δN atom of His429 forms an H-bond with the axial Cys436 thiolate whereas the eN atom forms an H-bond with the solvent and the side chain of Gln357. The amide of Gly438 forms the only other H-bond to the tetrahedral thiolate. Theoretical quantification of the histidine-thiolate interaction demonstrates a significant electron withdrawing effect on the heme iron. Comparisons of structures of class I-IV P450s demonstrate that either a phenylalanine or tryptophan is often found at the location corresponding to Phe429. Depending on the structure of the distal pocket heme, the residue at this location may or may not regulate the thermodynamic properties of the P450. Regardless, this residue appears to protect the thiolate from solvent, oxidation, protonations, and other deleterious reactions.
- Published
- 2014
19. Shortening spin–lattice relaxation using a copper-chelated lipid at low-temperatures – A magic angle spinning solid-state NMR study on a membrane-bound protein
- Author
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Ayyalusamy Ramamoorthy, Lucy Waskell, Marc A. Caporini, Sang Choul Im, and Kazutoshi Yamamoto
- Subjects
Nuclear and High Energy Physics ,Resolution (mass spectrometry) ,Biophysics ,Analytical chemistry ,chemistry.chemical_element ,Signal-To-Noise Ratio ,Biochemistry ,Article ,Magic angle spinning ,Animals ,Chelation ,Nuclear Magnetic Resonance, Biomolecular ,Chromatography, High Pressure Liquid ,Chelating Agents ,chemistry.chemical_classification ,Carbon Isotopes ,Nitrogen Isotopes ,Biomolecule ,Temperature ,Spin–lattice relaxation ,Membrane Proteins ,Nuclear magnetic resonance spectroscopy ,Condensed Matter Physics ,Lipids ,Copper ,Cytochromes b5 ,Solid-state nuclear magnetic resonance ,chemistry ,lipids (amino acids, peptides, and proteins) ,Rabbits - Abstract
Inherent low sensitivity of NMR spectroscopy has been a major disadvantage, especially to study biomolecules like membrane proteins. Recent studies have successfully demonstrated the advantages of performing solid-state NMR experiments at very low and ultralow temperatures to enhance the sensitivity. However, the long spin–lattice relaxation time, T 1 , at very low temperatures is a major limitation. To overcome this difficulty, we demonstrate the use of a copper-chelated lipid for magic angle spinning solid-state NMR measurements on cytochrome-b5 reconstituted in multilamellar vesicles. Our results on multilamellar vesicles containing as small as 0.5 mol% of a copper-chelated lipid can significantly shorten T 1 of protons, which can be used to considerably reduce the data collection time or to enhance the signal-to-noise ratio. We also monitored the effect of slow cooling on the resolution and sensitivity of 13 C and 15 N signals from the protein and 13 C signals from lipids.
- Published
- 2013
20. Cytochrome-P450–Cytochrome-b5 Interaction in a Membrane Environment Changes 15N Chemical Shift Anisotropy Tensors
- Author
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Shivani Ahuja, Ayyalusamy Ramamoorthy, Manoj Kumar Pandey, Rui Huang, Sang Choul Im, Lucy Waskell, and Subramanian Vivekanandan
- Subjects
Nitrogen Isotopes ,biology ,Chemistry ,Cytochrome P450 ,Nuclear magnetic resonance spectroscopy ,Amides ,Article ,Protein Structure, Tertiary ,Surfaces, Coatings and Films ,Crystallography ,Cytochromes b5 ,Membrane ,Cytochrome P-450 Enzyme System ,Cytochrome b5 ,Materials Chemistry ,biology.protein ,Animals ,Protein Interaction Domains and Motifs ,Rabbits ,Physical and Theoretical Chemistry ,Anisotropy ,Nuclear Magnetic Resonance, Biomolecular ,Peptide sequence ,Protein secondary structure ,Magnetic dipole–dipole interaction - Abstract
It has been well realized that the dependence of chemical shift anisotropy (CSA) tensors on the amino acid sequence, secondary structure, dynamics, and electrostatic interactions can be utilized in the structural and dynamic studies of proteins by NMR spectroscopy. In addition, CSA tensors could also be utilized to measure the structural interactions between proteins in a protein-protein complex. To this end, we report the experimentally measured backbone amide-(15)N CSA tensors for a membrane-bound 16.7 kDa full-length rabbit cytochrome-b5 (cytb5), in complexation with a 55.8 kDa microsomal rabbit cytochrome P450 2B4 (cytP4502B4). The (15)N-CSAs, determined using the (15)N CSA/(15)N-(1)H dipolar coupling transverse cross-correlated rates, for free cytb5 are compared with those for the cytb5 bound to cytP4502B4. An overall increase in backbone amide-(15)N transverse cross-correlated rates for the cytb5 residues in the cytb5-cytP450 complex is observed as compared to the free cytb5 residues. Due to fast spin-spin relaxation (T2) and subsequent broadening of the signals in the complex, we could measure amide-(15)N CSAs only for 48 residues of cytb5 as compared to 84 residues of free cytb5. We observed a change in (15)N CSA for most residues of cytb5 in the complex, as compared to free cytb5, suggesting a dynamic interaction between the oppositely charged surfaces of anionic cytb5 and cationic cytP450. The mean values of (15)N CSA determined for residues in helical, sheet, and turn regions of cytb5 in the complex are -184.5, -146.8, and -146.2 ppm, respectively, with an overall average value of -165.5 ppm (excluding the values from residues in more flexible termini). The measured CSA value for residues in helical conformation is slightly larger as compared to previously reported values. This may be attributed to the paramagnetic effect from Fe(III) of the heme in cytb5, which is similar to our previously reported values for the free cytb5.
- Published
- 2013
21. 1H, 13C and 15N resonance assignments for the full-length mammalian cytochrome b5 in a membrane environment
- Author
-
Subramanian Vivekanandan, Shivani Ahuja, Sang Choul Im, Lucy Waskell, and Ayyalusamy Ramamoorthy
- Subjects
Phosphorylcholine ,Chemistry ,Cholesterol ,medicine.medical_treatment ,Biochemistry ,Micelle ,Steroid ,Cell membrane ,chemistry.chemical_compound ,Membrane ,medicine.anatomical_structure ,Membrane protein ,Structural Biology ,Cytochrome b5 ,medicine - Abstract
Microsomal cytochrome b5 plays a key role in the oxidation of a variety of exogenous and endogenous compounds, including drugs, fatty acids, cholesterol and steroid hormones. To better understand its functional properties in a membrane mimic environment, we carried out high-resolution solution NMR studies. Here we report resonance assignments for full-length rabbit cytochrome b5 embedded in dodecylphosphocholine micelles.
- Published
- 2013
22. A Model of the Membrane-bound Cytochrome b5-Cytochrome P450 Complex from NMR and Mutagenesis Data
- Author
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Nataliya Popovych, Nicole Jahr, Stéphanie V. Le Clair, Subramanian Vivekanandan, Angela Bridges, Kazutoshi Yamamoto, Ravi Prakash Reddy Nanga, Sang Choul Im, Ronald Soong, Shivani Ahuja, Lucy Waskell, Jiadi Xu, Rui Huang, and Ayyalusamy Ramamoorthy
- Subjects
Models, Molecular ,Magnetic Resonance Spectroscopy ,Cytochrome ,Stereochemistry ,Molecular Sequence Data ,Heme ,Arginine ,Biochemistry ,Protein Structure, Secondary ,Substrate Specificity ,Electron Transport ,Electron transfer ,chemistry.chemical_compound ,Cytochrome P-450 Enzyme System ,Cytochrome b5 ,Animals ,Amino Acid Sequence ,Molecular Biology ,Binding Sites ,Sequence Homology, Amino Acid ,biology ,Chemistry ,Cell Biology ,Nuclear magnetic resonance spectroscopy ,Electron transport chain ,Protein Structure, Tertiary ,Kinetics ,Cytochromes b5 ,Structural biology ,Docking (molecular) ,Multiprotein Complexes ,Mutation ,Protein Structure and Folding ,Biocatalysis ,Mutagenesis, Site-Directed ,biology.protein ,Rabbits ,Oxidation-Reduction ,Protein Binding - Abstract
Microsomal cytochrome b5 (cytb5) is a membrane-bound protein that modulates the catalytic activity of its redox partner, cytochrome P4502B4 (cytP450). Here, we report the first structure of full-length rabbit ferric microsomal cytb5 (16 kDa), incorporated in two different membrane mimetics (detergent micelles and lipid bicelles). Differential line broadening of the cytb5 NMR resonances and site-directed mutagenesis data were used to characterize the cytb5 interaction epitope recognized by ferric microsomal cytP450 (56 kDa). Subsequently, a data-driven docking algorithm, HADDOCK (high ambiguity driven biomolecular docking), was used to generate the structure of the complex between cytP4502B4 and cytb5 using experimentally derived restraints from NMR, mutagenesis, and the double mutant cycle data obtained on the full-length proteins. Our docking and experimental results point to the formation of a dynamic electron transfer complex between the acidic convex surface of cytb5 and the concave basic proximal surface of cytP4502B4. The majority of the binding energy for the complex is provided by interactions between residues on the C-helix and β-bulge of cytP450 and residues at the end of helix α4 of cytb5. The structure of the complex allows us to propose an interprotein electron transfer pathway involving the highly conserved Arg-125 on cytP450 serving as a salt bridge between the heme propionates of cytP450 and cytb5. We have also shown that the addition of a substrate to cytP450 likely strengthens the cytb5-cytP450 interaction. This study paves the way to obtaining valuable structural, functional, and dynamic information on membrane-bound complexes.
- Published
- 2013
23. Probing the spontaneous membrane insertion of a tail-anchored membrane protein by sum frequency generation spectroscopy
- Author
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Khoi Tan Nguyen, Soong, Ronald, Sang-Choul Im, Waskell, Lucy, Ramamoorthy, Ayyalusamy, and Zhan Chen
- Subjects
Cytochrome b -- Structure ,Cytochrome b -- Chemical properties ,Gene mutations -- Analysis ,Lipid membranes -- Structure ,Lipid membranes -- Chemical properties ,Membrane proteins -- Structure ,Membrane proteins -- Chemical properties ,Vibrational spectra -- Analysis ,Chemistry - Abstract
Sum frequency generation (SFG) vibrational spectroscopy is used for examining the spontaneous membrane insertion process of cytochrome [b.sub.5] and its mutants. The results have correlated the nonfunctional property of a mutant cytochrome [b.sub.5] with its inability to insert into lipid bilayer and it is used as a potential rapid screening tool in measuring the topology of membrane proteins as well as interactions of biomolecules with lipid bilayers in situ.
- Published
- 2010
24. INEPT-based separated-local-field NMR spectroscopy: a unique approach to elucidate side-chain dynamics of membrane-associated proteins
- Author
-
Jiadi Xu, Soong, Ronald, Sang-Choul Im, Waskell, Lucy, and Ramamoorthy, Ayyalusamy
- Subjects
Lipid membranes -- Structure ,Lipid membranes -- Chemical properties ,Membrane proteins -- Structure ,Membrane proteins -- Chemical properties ,Nuclear magnetic resonance spectroscopy -- Usage ,Polarization (Electricity) -- Analysis ,Chemistry - Abstract
A solid-state NMR method, dipolar enhanced polarization transfer (DREPT), is described that has helped in determining the side-chain dynamics from membrane proteins in lipid bilayers. An appropriate model is used for determining the side-chain dynamics and providing additional information on the topology and function of a membrane protein in its native environment.
- Published
- 2010
25. Proton-evolved local-field solid-state NMR studies of cytochrome [b.sub.5] embedded in bicelles, revealing both structural and dynamical information
- Author
-
Soong, Ronald, Smith, Pieter E.S., Jiadi Xu, Yamamoto, Kazutoshi, Sang-Choul Im, Waskell, Lucy, and Ramamoorthy, Ayyalusamy
- Subjects
Cytochromes -- Chemical properties ,Cytochromes -- Electric properties ,Cytochromes -- Structure ,Magnetization -- Analysis ,Membrane proteins -- Structure ,Membrane proteins -- Chemical properties ,Membrane proteins -- Magnetic properties ,Nuclear magnetic resonance spectroscopy -- Usage ,Chemistry - Abstract
Two-dimensional proton-evolved local-field (2D PELF) pulse sequences are implemented by using either composite zero cross-polarization (COMPOZER-CP) or windowless isotropic mixing (WIM) for magnetization transfer. The results from magnetically aligned bicelles containing uniformly [super 15]N-labeled cytochrome [b.sub.5] have shown that the PELF-based experimental approaches have a major impact on solid-state NMR spectroscopy of membrane proteins and other membrane-associated molecules in magnetically aligned bicelles.
- Published
- 2010
26. Cytochrome b5 Activates the 17,20-Lyase Activity of Human Cytochrome P450 17A1 by Increasing the Coupling of NADPH Consumption to Androgen Production
- Author
-
Juilee Rege, Hwei Ming Peng, Richard J. Auchus, Lucy Waskell, Sang Choul Im, Adina F. Turcu, and Naw May Pearl
- Subjects
0301 basic medicine ,Models, Molecular ,medicine.medical_specialty ,medicine.drug_class ,Protein Conformation ,medicine.medical_treatment ,Biochemistry ,Article ,Steroid ,03 medical and health sciences ,Enzyme activator ,chemistry.chemical_compound ,Internal medicine ,Catalytic Domain ,Cytochrome b5 ,medicine ,Humans ,Enzyme Inhibitors ,Lyase activity ,NADPH-Ferrihemoprotein Reductase ,030102 biochemistry & molecular biology ,Allopregnanolone ,Cytochrome P450 reductase ,Steroid 17-alpha-Hydroxylase ,Androgen ,Recombinant Proteins ,Enzyme Activation ,030104 developmental biology ,Endocrinology ,Cytochromes b5 ,chemistry ,Amino Acid Substitution ,Pregnenolone ,Androgens ,Mutagenesis, Site-Directed ,Androstenes ,Mutant Proteins ,NADP ,medicine.drug - Abstract
Human cytochrome P450 17A1 is required for all androgen biosynthesis and is the target of abiraterone, a drug used widely to treat advanced prostate cancer. P450 17A1 catalyzes both 17-hydroxylation and subsequent 17,20-lyase reactions with pregnenolone, progesterone, and allopregnanolone. The presence of cytochrome b5 (b5) markedly stimulates the 17,20-lyase reaction, with little effect on 17-hydroxylation; however, the mechanism of this b5 effect is not known. We determined the influence of b5 on coupling efficiency-defined as the ratio of product formation to NADPH consumption-in a reconstituted system using these 3 pairs of substrates for the 2 reactions. Rates of NADPH consumption ranged from 4 to 13 nmol/min/nmol P450 with wild-type P450 17A1. For the 17-hydroxylase reaction, progesterone oxidation was the most tightly coupled (∼50%) and negligibly changed upon addition of b5. Rates of NADPH consumption were similar for the 17-hydroxylase and corresponding 17,20-lyase reactions for each steroid series, and b5 only slightly increased NADPH consumption. For the 17,20-lyase reactions, b5 markedly increased product formation and coupling in parallel with all substrates, from 6% to 44% with the major substrate 17-hydroxypregnenolone. For the naturally occurring P450 17A1 mutations E305G and R347H, which impair 17,20-lyase activity, b5 failed to rescue the poor coupling with 17-hydroxypregnenolone (2-4%). When the conserved active-site threonine was mutated to alanine (T306A), both the activity and coupling were markedly decreased with all substrates. We conclude that b5 stimulation of the 17,20-lyase reaction primarily derives from more efficient use of NADPH for product formation rather than side products.
- Published
- 2016
27. Mutants of Cytochrome P450 Reductase Lacking Either Gly-141 or Gly-143 Destabilize Its FMN Semiquinone*
- Author
-
Mohammad Mahfuzul Haque, Jung-Ja P. Kim, Freeborn Rwere, Chuanwu Xia, Sang Choul Im, Dennis J. Stuehr, and Lucy Waskell
- Subjects
0301 basic medicine ,Models, Molecular ,Cytochrome ,Semiquinone ,Stereochemistry ,Flavin Mononucleotide ,Protein Conformation ,Glycine ,Flavoprotein ,Flavin group ,Photochemistry ,Biochemistry ,Electron Transport ,03 medical and health sciences ,Cytochrome P-450 Enzyme System ,Oxidoreductase ,Animals ,Point Mutation ,Amino Acid Sequence ,Molecular Biology ,NADPH-Ferrihemoprotein Reductase ,Sequence Deletion ,chemistry.chemical_classification ,030102 biochemistry & molecular biology ,biology ,Chemistry ,Mutagenesis ,Cytochrome P450 reductase ,Cytochrome P450 ,Cytochromes c ,Cell Biology ,Rats ,030104 developmental biology ,Protein Synthesis and Degradation ,biology.protein ,Mutagenesis, Site-Directed ,Oxidation-Reduction ,NADP - Abstract
NADPH-cytochrome P450 oxidoreductase transfers electrons from NADPH to cytochromes P450 via its FAD and FMN. To understand the biochemical and structural basis of electron transfer from FMN-hydroquinone to its partners, three deletion mutants in a conserved loop near the FMN were characterized. Comparison of oxidized and reduced wild type and mutant structures reveals that the basis for the air stability of the neutral blue semiquinone is protonation of the flavin N5 and strong H-bond formation with the Gly-141 carbonyl. The ΔGly-143 protein had moderately decreased activity with cytochrome P450 and cytochrome c. It formed a flexible loop, which transiently interacts with the flavin N5, resulting in the generation of both an unstable neutral blue semiquinone and hydroquinone. The ΔGly-141 and ΔG141/E142N mutants were inactive with cytochrome P450 but fully active in reducing cytochrome c. In the ΔGly-141 mutants, the backbone amide of Glu/Asn-142 forms an H-bond to the N5 of the oxidized flavin, which leads to formation of an unstable red anionic semiquinone with a more negative potential than the hydroquinone. The semiquinone of ΔG141/E142N was slightly more stable than that of ΔGly-141, consistent with its crystallographically demonstrated more rigid loop. Nonetheless, both ΔGly-141 red semiquinones were less stable than those of the corresponding loop in cytochrome P450 BM3 and the neuronal NOS mutant (ΔGly-810). Our results indicate that the catalytic activity of cytochrome P450 oxidoreductase is a function of the length, sequence, and flexibility of the 140s loop and illustrate the sophisticated variety of biochemical mechanisms employed in fine-tuning its redox properties and function.
- Published
- 2016
28. Reconstitution of the Cytb5-CytP450 Complex in Nanodiscs for Structural Studies using NMR Spectroscopy
- Author
-
Ayyalusamy Ramamoorthy, Meng Zhang, Sang Choul Im, Rui Huang, Rose Ackermann, Anna Schwendeman, and Lucy Waskell
- Subjects
0301 basic medicine ,Magnetic Resonance Spectroscopy ,Cytochrome ,Size-exclusion chromatography ,Peptide ,Nanotechnology ,010402 general chemistry ,01 natural sciences ,Catalysis ,Protein–protein interaction ,03 medical and health sciences ,0302 clinical medicine ,Cytochrome P-450 Enzyme System ,Nanodisc ,chemistry.chemical_classification ,biology ,SUPERFAMILY ,General Chemistry ,Nuclear magnetic resonance spectroscopy ,General Medicine ,Combinatorial chemistry ,0104 chemical sciences ,Nanostructures ,Membrane ,030104 developmental biology ,Cytochromes b5 ,chemistry ,biology.protein ,Chromatography, Gel ,030217 neurology & neurosurgery - Abstract
Cytochrome P450s (P450s) are a superfamily of enzymes responsible for the catalysis of a wide range of substrates. Dynamic interactions between full-length membrane-bound P450 and its redox partner cytochrome b5 (cytb5 ) have been found to be important for the enzymatic activity of P450. However, the stability of the circa 70 kDa membrane-bound complex in model membranes renders high-resolution structural NMR studies particularly difficult. To overcome these challenges, reconstitution of the P450-cytb5 complex in peptide-based nanodiscs, containing no detergents, has been demonstrated, which are characterized by size exclusion chromatography and NMR spectroscopy. In addition, NMR experiments are used to identify the binding interface of the P450-cytb5 complex in the nanodisc. This is the first successful demonstration of a protein-protein complex in a nanodisc using NMR structural studies and should be useful to obtain valuable structural information on membrane-bound protein complexes.
- Published
- 2016
29. Kinetics of oxidation of benzphetamine by compounds I of cytochrome P450 2B4 and its mutants
- Author
-
Xin Sheng, Haoming Zhang, Sang-Choul Im, Horner, John H., Waskell, Lucy, Hollenberg, Paul F., and Newcomb, Martin
- Subjects
Cytochrome P-450 -- Structure ,Cytochrome P-450 -- Chemical properties ,Enzyme binding -- Analysis ,Mutation (Biology) -- Analysis ,Oxidation-reduction reaction -- Analysis ,Photolysis -- Analysis ,Porphyrins -- Chemical properties ,Porphyrins -- Structure ,Chemistry - Abstract
Iron(IV)-oxo porphyrin radical cations (Compound I) of the mammalian hepatic P450 enzyme CYP2B4 and three mutants are prepared by laser flash photolysis of the Compound II species that, in turn, are prepared by reaction of the resting enzymes with peroxynitrite. The studies have shown that substrate binding constants for P450 Compound I are important for controlling overall rates of oxidation reactions and the intrinsic reactivities of Compounds I from various P450 enzymes are comparable.
- Published
- 2009
30. Determination of 15N Chemical Shift Anisotropy from a Membrane-Bound Protein by NMR Spectroscopy
- Author
-
Manoj Kumar Pandey, Kumar Pichumani, Shivani Ahuja, Sang Choul Im, Lucy Waskell, Ayyalusamy Ramamoorthy, and Subramanian Vivekanandan
- Subjects
chemistry.chemical_classification ,Nitrogen Isotopes ,Globular protein ,Relaxation (NMR) ,Heme ,Nuclear magnetic resonance spectroscopy ,Ferric Compounds ,Article ,Surfaces, Coatings and Films ,Turn (biochemistry) ,chemistry.chemical_compound ,Crystallography ,Cytochromes b5 ,chemistry ,Membrane protein ,Materials Chemistry ,Animals ,Rabbits ,Physical and Theoretical Chemistry ,Anisotropy ,Nuclear Magnetic Resonance, Biomolecular ,Protein secondary structure - Abstract
Chemical shift anisotropy (CSA) tensors are essential in the structural and dynamic studies of proteins using NMR spectroscopy. Results from relaxation studies in biomolecular solution and solid-state NMR experiments on aligned samples are routinely interpreted using well-characterized CSA tensors determined from model compounds. Since CSA tensors, particularly the (15)N CSA, highly depend on a number of parameters including secondary structure, electrostatic interaction, and the amino acid sequence, there is a need for accurately determined CSA tensors from proteins. In this study, we report the backbone amide-(15)N CSA tensors for a 16.7-kDa membrane-bound and paramagnetic-heme containing protein, rabbit Cytochrome b(5) (cytb(5)), determined using the (15)N CSA/(15)N-(1)H dipolar transverse cross-correlation rates. The mean values of (15)N CSA determined for residues in helical, sheet, and turn regions are -187.9, -166.0, and -161.1 ppm, respectively, with an overall average value of -171.7 ppm. While the average CSA value determined from this study is in good agreement with previous solution NMR experiments on small globular proteins, the CSA value determined for residues in helical conformation is slightly larger, which may be attributed to the paramagnetic effect from Fe(III) of the heme unit in cytb(5). However, like in previous solution NMR studies, the CSA values reported in this study are larger than the values measured from solid-state NMR experiments. We believe that the CSA parameters reported in this study will be useful in determining the structure, dynamics, and orientation of proteins, including membrane proteins, using NMR spectroscopy.
- Published
- 2012
31. Resonance Raman studies of cytochrome P450 2B4 in its interactions with substrates and redox partners
- Author
-
Mak, Piotr J., Sang-Choul Im, Haoming Zhang, Waskell, Lucy A., and Kincaid, James R.
- Subjects
Cresol -- Chemical properties ,Cytochrome P-450 -- Structure ,Cytochrome P-450 -- Chemical properties ,Protein binding -- Analysis ,Raman spectroscopy -- Usage ,Biological sciences ,Chemistry - Abstract
A detailed resonance Raman studies of the heme structure of P450 2B4 in the substrate-free form and bound to either of two different substrates, benzphetamine (BZ) or butylated hydroxytoluene (BHT) is reported. The observation based on RR results suggest that the combination of benzphetamine and cytochrome (cyt [b.sub.5]) binding produce a synergy leading to unique active site structural changes when both are bound.
- Published
- 2008
32. Conformational Changes of NADPH-Cytochrome P450 Oxidoreductase Are Essential for Catalysis and Cofactor Binding
- Author
-
Chuanwu Xia, Charles B. Kasper, Vivian Choi, Djemel Hamdane, Naw May Pearl, Sang Choul Im, Jung-Ja P. Kim, Haoming Zhang, Anna L. Shen, and Lucy Waskell
- Subjects
Flavin Mononucleotide ,Stereochemistry ,Mutation, Missense ,Flavin mononucleotide ,Flavoprotein ,Flavin group ,Crystallography, X-Ray ,Biochemistry ,Protein Structure, Secondary ,Electron Transport ,chemistry.chemical_compound ,Oxidoreductase ,Animals ,Molecular Biology ,NADPH-Ferrihemoprotein Reductase ,chemistry.chemical_classification ,Cofactor binding ,biology ,Cytochrome P450 reductase ,Cell Biology ,Electron transport chain ,Protein Structure, Tertiary ,Rats ,Amino Acid Substitution ,chemistry ,Enzymology ,biology.protein ,NADPH binding ,Oxidation-Reduction ,NADP ,Protein Binding - Abstract
The crystal structure of NADPH-cytochrome P450 reductase (CYPOR) implies that a large domain movement is essential for electron transfer from NADPH via FAD and FMN to its redox partners. To test this hypothesis, a disulfide bond was engineered between residues Asp(147) and Arg(514) in the FMN and FAD domains, respectively. The cross-linked form of this mutant protein, designated 147CC514, exhibited a significant decrease in the rate of interflavin electron transfer and large (≥90%) decreases in rates of electron transfer to its redox partners, cytochrome c and cytochrome P450 2B4. Reduction of the disulfide bond restored the ability of the mutant to reduce its redox partners, demonstrating that a conformational change is essential for CYPOR function. The crystal structures of the mutant without and with NADP(+) revealed that the two flavin domains are joined by a disulfide linkage and that the relative orientations of the two flavin rings are twisted ∼20° compared with the wild type, decreasing the surface contact area between the two flavin rings. Comparison of the structures without and with NADP(+) shows movement of the Gly(631)-Asn(635) loop. In the NADP(+)-free structure, the loop adopts a conformation that sterically hinders NADP(H) binding. The structure with NADP(+) shows movement of the Gly(631)-Asn(635) loop to a position that permits NADP(H) binding. Furthermore, comparison of these mutant and wild type structures strongly suggests that the Gly(631)-Asn(635) loop movement controls NADPH binding and NADP(+) release; this loop movement in turn facilitates the flavin domain movement, allowing electron transfer from FMN to the CYPOR redox partners.
- Published
- 2011
33. Stabilization and spectroscopic characterization of the dioxygen complex of wild-type cytochrome P4502B4 (CYP2B4) and its distal side E301Q, T302A and proximal side F429H mutants at subzero temperatures
- Author
-
Michael Tarasev, Ryan D. Kinloch, Masanori Sono, Roshan Perera, Sang Choul Im, John H. Dawson, Haoming Zhang, and Lucy Waskell
- Subjects
Models, Molecular ,Cytochrome ,Stereochemistry ,Iron ,Biophysics ,Heme ,Biochemistry ,Article ,Substrate Specificity ,Analytical Chemistry ,chemistry.chemical_compound ,Catalytic Domain ,Organometallic Compounds ,medicine ,Animals ,Cytochrome P450 Family 2 ,Molecular Biology ,chemistry.chemical_classification ,biology ,Transition metal dioxygen complex ,Circular Dichroism ,Wild type ,Benzphetamine ,Cytochrome P450 ,Monooxygenase ,Protein Structure, Tertiary ,Amino acid ,Cold Temperature ,Oxygen ,Crystallography ,Amino Acid Substitution ,chemistry ,Spectrophotometry ,Mutation ,biology.protein ,Aryl Hydrocarbon Hydroxylases ,Rabbits ,Protein Binding ,medicine.drug - Abstract
Mammalian cytochrome P450 2B4 (CYP2B4) is a phenobarbital-inducible rabbit hepatic monooxygenase that catalyzes the N-demethylation of benzphetamine and metabolism of numerous other compounds. To probe the interactions of the heme environment and bound benzphetamine with the dioxygen (O2) complex of CYP2B4, homogeneous O2 complexes of the wild-type enzyme and three mutants at sites of conserved amino acids, two on the heme distal side (T302A and E301Q) and one on the proximal side (F429H), have been prepared and stabilized at ~−50 °C in mixed solvents (60–70% v/v glycerol). We report that the magnetic circular dichroism and electronic absorption spectra of wild-type oxyferrous CYP2B4, in the presence and absence of substrate, are quite similar to those of the dioxygen complex of bacterial cytochrome P450-CAM (CYP101). However, the oxyferrous complexes of the T302A and E301Q CYP2B4 mutants have significantly perturbed electronic structure (~ 4 nm and ~ 3 nm red-shifted Soret features, respectively) compared to that of the wild-type oxyferrous complex. On the other hand, the heme proximal side mutant, CYP2B4 F429H, undergoes relatively facile conversion to a partially (~ 50%) denatured (P420) form upon reduction. The structural changes in the heme pocket environments of the CYP2B4 mutants that lead to the spectroscopic distinctions reported herein can be related to the differences in oxidation activities of wild-type CYP2B4 and its E301Q, T302A and F429H mutants.
- Published
- 2011
34. Proton-Evolved Local-Field Solid-State NMR Studies of Cytochrome b5 Embedded in Bicelles, Revealing both Structural and Dynamical Information
- Author
-
Lucy Waskell, Sang Choul Im, Ayyalusamy Ramamoorthy, Jiadi Xu, Pieter E. S. Smith, Kazutoshi Yamamoto, and Ronald Soong
- Subjects
inorganic chemicals ,Molecular Sequence Data ,Heme ,Molecular Dynamics Simulation ,Model lipid bilayer ,Biochemistry ,Protein Structure, Secondary ,Article ,Catalysis ,Electron Transport ,Quantitative Biology::Subcellular Processes ,Magnetics ,Molecular dynamics ,Colloid and Surface Chemistry ,Nuclear magnetic resonance ,Animals ,Amino Acid Sequence ,Magnetization transfer ,Nuclear Magnetic Resonance, Biomolecular ,Chemistry ,Protein dynamics ,Cell Membrane ,Water ,General Chemistry ,Nuclear magnetic resonance spectroscopy ,Protein Structure, Tertiary ,Cytochromes b5 ,Solubility ,Solid-state nuclear magnetic resonance ,Heteronuclear molecule ,Residual dipolar coupling ,Chemical physics ,Mutation ,Rabbits ,Protons - Abstract
Structural biology of membrane proteins has rapidly evolved into a new frontier of science. Although solving the structure of a membrane protein with atomic-level resolution is still a major challenge, separated local field (SLF) NMR spectroscopy has become an invaluable tool in obtaining structural images of membrane proteins under physiological conditions. Recent studies have demonstrated the use of rotating-frame SLF techniques to accurately measure strong heteronuclear dipolar couplings between directly bonded nuclei. However, in these experiments, all weak dipolar couplings are suppressed. On the other hand, weak heteronuclear dipolar couplings can be measured using laboratory-frame SLF experiments, but only at the expense of spectral resolution for strongly dipolar coupled spins. In the present study, we implemented two-dimensional proton-evolved local-field (2D PELF) pulse sequences using either composite zero cross-polarization (COMPOZER-CP) or windowless isotropic mixing (WIM) for magnetization transfer. These PELF sequences can be used for the measurement of a broad range of heteronuclear dipolar couplings, allowing for a complete mapping of protein dynamics in a lipid bilayer environment. Experimental results from magnetically aligned bicelles containing uniformly (15)N-labeled cytochrome b(5) are presented and theoretical analyses of the new PELF sequences are reported. Our results suggest that the PELF-based experimental approaches will have a profound impact on solid-state NMR spectroscopy of membrane proteins and other membrane-associated molecules in magnetically aligned bicelles.
- Published
- 2010
35. Structure and Function of an NADPH-Cytochrome P450 Oxidoreductase in an Open Conformation Capable of Reducing Cytochrome P450
- Author
-
Sang Choul Im, Djemel Hamdane, Chuanwu Xia, Lucy Waskell, Haoming Zhang, and Jung-Ja P. Kim
- Subjects
Flavin Mononucleotide ,Protein Conformation ,Stereochemistry ,Molecular Conformation ,Flavin mononucleotide ,Electrons ,Flavin group ,Biochemistry ,chemistry.chemical_compound ,Protein structure ,Oxidoreductase ,Flavins ,Animals ,Molecular Biology ,NADPH-Ferrihemoprotein Reductase ,chemistry.chemical_classification ,Enzyme Catalysis and Regulation ,biology ,C-terminus ,Mutagenesis ,Benzphetamine ,Cytochrome P450 ,Cell Biology ,Protein Structure, Tertiary ,Rats ,Kinetics ,chemistry ,Mutation ,biology.protein ,Oxidation-Reduction ,Linker ,Gene Deletion - Abstract
NADPH-cytochrome P450 oxidoreductase (CYPOR) catalyzes the transfer of electrons to all known microsomal cytochromes P450. A CYPOR variant, with a 4-amino acid deletion in the hinge connecting the FMN domain to the rest of the protein, has been crystallized in three remarkably extended conformations. The variant donates an electron to cytochrome P450 at the same rate as the wild-type, when provided with sufficient electrons. Nevertheless, it is defective in its ability to transfer electrons intramolecularly from FAD to FMN. The three extended CYPOR structures demonstrate that, by pivoting on the C terminus of the hinge, the FMN domain of the enzyme undergoes a structural rearrangement that separates it from FAD and exposes the FMN, allowing it to interact with its redox partners. A similar movement most likely occurs in the wild-type enzyme in the course of transferring electrons from FAD to its physiological partner, cytochrome P450. A model of the complex between an open conformation of CYPOR and cytochrome P450 is presented that satisfies mutagenesis constraints. Neither lengthening the linker nor mutating its sequence influenced the activity of CYPOR. It is likely that the analogous linker in other members of the diflavin family functions in a similar manner.
- Published
- 2009
36. Kinetics of Oxidation of Benzphetamine by Compounds I of Cytochrome P450 2B4 and Its Mutants
- Author
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Paul F. Hollenberg, Lucy Waskell, Sang Choul Im, Haoming Zhang, Xin Sheng, John H. Horner, and Martin Newcomb
- Subjects
Models, Molecular ,Spectrometry, Mass, Electrospray Ionization ,Archaeal Proteins ,Kinetics ,Photochemistry ,Biochemistry ,Redox ,Article ,Catalysis ,chemistry.chemical_compound ,Colloid and Surface Chemistry ,Cytochrome P-450 Enzyme System ,Nitration ,medicine ,Reactivity (chemistry) ,Enzyme kinetics ,Cytochrome P450 Family 2 ,Chromatography, High Pressure Liquid ,Photolysis ,Benzphetamine ,General Chemistry ,Photochemical Processes ,Porphyrin ,chemistry ,Flash photolysis ,Spectrophotometry, Ultraviolet ,Aryl Hydrocarbon Hydroxylases ,Oxidation-Reduction ,medicine.drug - Abstract
Cytochromes P450 are ubiquitous heme-containing enzymes that catalyze a wide range of reactions in nature including many oxidation reactions. The active oxidant species in P450 enzymes are widely thought to be iron(IV)-oxo porphyrin radical cations, termed Compound I species, but these intermediates have not been observed under turnover conditions. We prepared Compounds I of the mammalian hepatic P450 enzyme CYP2B4 and three mutants (E301Q, T302A, and F429H) by laser flash photolysis of the Compound II species that, in turn, were prepared by reaction of the resting enzymes with peroxynitrite. The PN treatment resulted in a small amount of nitration of the P450 as determined by mass spectrometry but no change in reactivity of the P450 in a test reaction. CYP2B4 Compound I oxidized benzphetamine to norbenzphetamine in high yield in bulk studies. In direct kinetic studies of benzphetamine oxidations, Compounds I displayed saturation kinetics with similar binding equilibrium constants (K(bind)) for each. The first-order oxidation rate constants (k(ox)) were comparable for Compounds I of CYP2B4, the E301Q mutant, and the T302A mutant, whereas the k(ox) for Compound I of the F429H mutant was reduced by a factor of 2. CYP119 Compound I, studied for comparison purposes, reacted with benzphetamine with a binding constant that was nearly an order of magnitude smaller than that of CYP2B4 but a rate constant that was similar. Substrate binding constants for P450 Compound I are important for controlling overall rates of oxidation reactions, and the intrinsic reactivities of Compounds I from various P450 enzymes are comparable.
- Published
- 2009
37. Characterization and calculation of a cytochrome c-cytochrome b5 complex using NMR data
- Author
-
Deep, Shashank, Sang-Choul Im, Zuiderweg, Erik R.P., and Waskell, Lucy
- Subjects
Nuclear magnetic resonance -- Analysis ,Cytochrome c -- Research ,Protein research ,Biological sciences ,Chemistry - Abstract
The cross-saturation transfer NMR experiments performed with H and N-enriched cytochrome c (cyt c) helped to identify the binding site for Bovine cytochrome b5 (cyt b5) on horse cytochrome c (cyt c). It was seen that presence of different amino acids at the protein-protein interface and the dissimilar assumptions in the calculations was the main cause for the non identical interfaces.
- Published
- 2005
38. Bicelle-Enabled Structural Studies on a Membrane-Associated Cytochrome b5by Solid-State MAS NMR Spectroscopy
- Author
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Zhehong Gan, Lucy Waskell, Ayyalusamy Ramamoorthy, Ulrich H.N. Dürr, Sang Choul Im, and Jiadi Xu
- Subjects
Chemistry ,Vesicle ,Analytical chemistry ,General Medicine ,General Chemistry ,Nuclear magnetic resonance spectroscopy ,Model lipid bilayer ,Catalysis ,Transmembrane protein ,Cell membrane ,medicine.anatomical_structure ,Membrane ,Protein structure ,Membrane protein ,medicine ,Biophysics - Abstract
membrane proteins still remain a great challenge, mainly because of the difficulty in finding a well-behaved model membrane. The use of multi-lamellar vesicles containing a transmembrane protein could enable the application of solidstate NMR spectroscopic techniques, but they are not usually suitable, as membrane proteins containing large soluble domains may not fold well to result in high-resolution spectra. Obtaining high-resolution spectra is a mandatory first step in solving the protein structure using NMR spectroscopy. In this study we demonstrate that bicelles [2] are suitable to overcome
- Published
- 2008
39. Characterization of the Microsomal Cytochrome P450 2B4 O2 Activation Intermediates by Cryoreduction and Electron Paramagnetic Resonance
- Author
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Sang Choul Im, Roman Davydov, Reza Razeghifard, Lucy Waskell, and Brian M. Hoffman
- Subjects
biology ,Chemistry ,Annealing (metallurgy) ,Analytical chemistry ,Active site ,Cytochrome P450 ,Biochemistry ,law.invention ,Crystallography ,chemistry.chemical_compound ,Microsomal p450 ,law ,biology.protein ,Microsome ,Butylated hydroxytoluene ,Electron paramagnetic resonance ,Ternary operation - Abstract
The oxy−ferrous complex of cytochrome P450 2B4 (2B4) has been prepared at −40 °C with and without bound substrate [butylated hydroxytoluene (BHT)] and radiolytically one-electron cryoreduced at 77 K. Electron paramagnetic resonance (EPR) shows that in both cases the observed product of cryoreduction is the hydroperoxo−ferriheme species, indicating that the microsomal P450 contains an efficient distal-pocket proton-delivery network. In the absence of substrate, two distinct hydroperoxo−ferriheme signals are observed, reflecting the presence of two major conformational substates in the oxy−ferrous precursor. Only one species is observed when BHT is bound, indicating a more ordered active site. BHT binding also changes the g-tensor components of the hydroperoxo−ferric 2B4 intermediate, indicating that the substrate modulates the properties of this intermediate. Step annealing the cryoreduced ternary 2B4 complex at ≥175 K causes the loss of hydroperoxo−ferric 2B4 and the parallel appearance of high-spin ferri...
- Published
- 2008
40. Resonance Raman Studies of Cytochrome P450 2B4 in Its Interactions with Substrates and Redox Partners
- Author
-
Haoming Zhang, Lucy Waskell, James R. Kincaid, Sang-Choul Im, and Piotr J. Mak
- Subjects
Heme binding ,Stereochemistry ,Spectrum Analysis, Raman ,Ferric Compounds ,Biochemistry ,chemistry.chemical_compound ,medicine ,Animals ,Cytochrome P450 Family 2 ,Heme ,NADPH-Ferrihemoprotein Reductase ,Manganese ,biology ,Cytochrome b ,Chemistry ,Benzphetamine ,Cytochrome P450 reductase ,Cytochrome P450 ,Active site ,Substrate (chemistry) ,Butylated Hydroxytoluene ,Cytochromes b5 ,biology.protein ,Aryl Hydrocarbon Hydroxylases ,Rabbits ,Oxidation-Reduction ,medicine.drug - Abstract
Resonance Raman studies of P450 2B4 are reported for the substrate-free form and when bound to the substrates, benzphetamine (BZ) or butylated hydroxytoluene (BHT), the latter representing a substrate capable of inducing an especially effective conversion to the high-spin state. In addition to studies of the ferric resting state, spectra are acquired for the ferrous CO ligated form. Importantly, for the first time, the RR technique is effectively applied to interrogate the changes in active site structure induced by binding of cytochrome P450 reductase (CPR) and Mn(III) cytochrome b 5 (Mn cyt b 5); the manganese derivative of cyt b 5 was employed to avoid spectroscopic interferences. The results, consistent with early work on mammalian P450s, demonstrate that substrate structure has minimal effects on heme structure or the FeCO fragment of the ferrous CO derivatives. Similarly, the data indicate that the protein is flexible and that substrate binding does not exert significant strain on the heme peripheral groups, in contrast to P450 cam, where substantial effects on heme peripheral groups are seen. However, significant differences are observed in the RR spectra of P450 2B4 when bound with the different redox partners, indicating that the heme structure is clearly sensitive to perturbations near the proximal heme binding site. The most substantial changes are displacements of the peripheral vinyl groups toward planarity with the heme macrocycle by cyt b 5 but away from planarity by CPR. These changes can have an impact on heme reduction potential. Most interestingly, these RR results support an earlier observation that the combination of benzphetamine and cyt b 5 binding produce a synergy leading to unique active site structural changes when both are bound.
- Published
- 2008
41. Cytochrome b5 Inhibits Electron Transfer from NADPH-Cytochrome P450 Reductase to Ferric Cytochrome P450 2B4
- Author
-
Djemel Hamdane, Sang Choul Im, Haoming Zhang, and Lucy Waskell
- Subjects
Cytochrome ,Reductase ,Biochemistry ,Catalysis ,Electron Transport ,Electron transfer ,Cytochrome b5 ,medicine ,Animals ,Cytochrome P450 Family 2 ,Molecular Biology ,NADPH-Ferrihemoprotein Reductase ,Manganese ,Binding Sites ,biology ,Chemistry ,Cytochrome c ,Mutagenesis ,Cytochrome P450 ,Cell Biology ,Molecular biology ,Recombinant Proteins ,Cytochromes b5 ,Mutagenesis, Site-Directed ,biology.protein ,Ferric ,Aryl Hydrocarbon Hydroxylases ,Protein Binding ,medicine.drug - Abstract
Experiments demonstrating that cytochrome (cyt) b5 inhibits the activity of cytochrome P450 2B4 (cyt P450 2B4) at higher concentrations suggested that cyt b5 was occupying the cyt P450 reductase-binding site on cyt P450 2B4 and preventing the reduction of ferric cyt P450 (Zhang, H., Im, S.-C., and Waskell, L. (2007) J. Biol. Chem. 282, 29766-29776). In this work experiments were undertaken with manganese-containing cyt b5 (Mn-cyt b5) to test this hypothesis. Because Mn-cyt b5 does not undergo oxidation state changes under our experimental conditions, interpretation of the experimental results was unambiguous. The rate of electron transfer from cyt P450 reductase to ferric cyt P450 2B4 was decreased by Mn-cyt b5 in a concentration-dependent manner. Moreover, reduction of cyt P450 2B4 by cyt P450 reductase was incomplete in the presence of Mn-cyt b5. At a Mn-cyt b(5):cyt P450 2B4:cyt P450 reductase molar ratio of 5:1:1, the rate of reduction of ferric cyt P450 was decreased by 10-fold, and only 30% of the cyt P450 was reduced, whereas 70% remained oxidized. It could be demonstrated that Mn-cyt b5 had its effect by acting on cyt P450, not the reductase, because the reduction of cyt c by cyt P450 reductase in the presence of Mn-cyt b5 was not effected. Furthermore, under steady-state conditions in the cyt P450 reconstituted system, Mn-cyt b5, which lacks the ability to reduce oxyferrous cyt P450 2B4, was unable to stimulate the activity of cyt P450. Mn-cyt b5 only inhibited the cyt P450 2B4 activity. In conjunction with site-directed mutagenesis studies and experiments that strongly suggested that cyt b5 competed with cyt P450 reductase for binding to cyt P450, the current investigation demonstrates unequivocally that cyt b5 inhibits the activity of cyt P450 2B4 by preventing cyt P450 reductase from binding to cyt P450, a prerequisite for electron transfer from cyt P450 reductase to cyt P450 and catalysis.
- Published
- 2008
42. Insights into the Role of Substrates on the Interaction between Cytochrome b5 and Cytochrome P450 2B4 by NMR
- Author
-
Rui Huang, Ayyalusamy Ramamoorthy, Lucy Waskell, Meng Zhang, Shivani Ahuja, Sang Choul Im, and Stéphanie V. Le Clair
- Subjects
Models, Molecular ,Protein Conformation ,Population ,Plasma protein binding ,Sodium Chloride ,environment and public health ,Article ,Substrate Specificity ,Protein structure ,Cytochrome b5 ,Cytochrome P450 Family 2 ,education ,Nuclear Magnetic Resonance, Biomolecular ,education.field_of_study ,Multidisciplinary ,biology ,Chemistry ,Osmolar Concentration ,Substrate (chemistry) ,Cytochrome P450 ,enzymes and coenzymes (carbohydrates) ,Cytochromes b5 ,Catalytic cycle ,Biochemistry ,Ionic strength ,embryonic structures ,cardiovascular system ,Biophysics ,biology.protein ,Aryl Hydrocarbon Hydroxylases ,Protein Binding - Abstract
Mammalian cytochrome b5 (cyt b5) is a membrane-bound protein capable of donating an electron to cytochrome P450 (P450) in the P450 catalytic cycle. The interaction between cyt b5 and P450 has been reported to be affected by the substrates of P450; however, the mechanism of substrate modulation on the cyt b5-P450 complex formation is still unknown. In this study, the complexes between full-length rabbit cyt b5 and full-length substrate-free/substrate-bound cytochrome P450 2B4 (CYP2B4) are investigated using NMR techniques. Our findings reveal that the population of complexes is ionic strength dependent, implying the importance of electrostatic interactions in the complex formation process. The observation that the cyt b5-substrate-bound CYP2B4 complex shows a weaker dependence on ionic strength than the cyt b5-substrate-free CYP2B4 complex suggests the presence of a larger fraction of steoreospecific complexes when CYP2B4 is substrate-bound. These results suggest that a CYP2B4 substrate likely promotes specific interactions between cyt b5 and CYP2B4. Residues D65, V66, T70, D71 and A72 are found to be involved in specific interactions between the two proteins due to their weak response to ionic strength change. These findings provide insights into the mechanism underlying substrate modulation on the cyt b5-P450 complexation process.
- Published
- 2015
43. Temperature-resistant bicelles for structural studies by solid-state NMR spectroscopy
- Author
-
Paige Pearcy, Lucy Waskell, Changsu Yu, Ayyalusamy Ramamoorthy, Dong Kuk Lee, Sang Choul Im, and Kazutoshi Yamamoto
- Subjects
Chemistry ,Protein Conformation ,Temperature ,Membrane Proteins ,Surfaces and Interfaces ,Model lipid bilayer ,Condensed Matter Physics ,Micelle ,Folding (chemistry) ,Transmembrane domain ,Crystallography ,Membrane protein ,Solid-state nuclear magnetic resonance ,Electrochemistry ,Biophysics ,General Materials Science ,Spectroscopy ,Integral membrane protein ,Nuclear Magnetic Resonance, Biomolecular ,Micelles - Abstract
Three-dimensional structure determination of membrane proteins is important to fully understand their biological functions. However, obtaining a high-resolution structure has been a major challenge mainly due to the difficulties in retaining the native folding and function of membrane proteins outside of the cellular membrane environment. These challenges are acute if the protein contains a large soluble domain, as it needs bulk water unlike the transmembrane domains of an integral membrane protein. For structural studies on such proteins either by nuclear magnetic resonance (NMR) spectroscopy or X-ray crystallography, bicelles have been demonstrated to be superior to conventional micelles, yet their temperature restrictions attributed to their thermal instabilities are a major disadvantage. Here, we report an approach to overcome this drawback through searching for an optimum combination of bicellar compositions. We demonstrate that bicelles composed of 1,2-didecanoyl-sn-glycero-3-phosphocholine (DDPC) and 1,2-diheptanoyl-sn-glycero-3-phosphocholin (DHepPC), without utilizing additional stabilizing chemicals, are quite stable and are resistant to temperature variations. These temperature-resistant bicelles have a robust bicellar phase and magnetic alignment over a broad range of temperatures, between -15 and 80 °C, retain the native structure of a membrane protein, and increase the sensitivity of solid-state NMR experiments performed at low temperatures. Advantages of two-dimensional separated-local field (SLF) solid-state NMR experiments at a low temperature are demonstrated on magnetically aligned bicelles containing an electron carrier membrane protein, cytochrome b5. Morphological information on different DDPC-based bicellar compositions, varying q ratio/size, and hydration levels obtained from (31)P NMR experiments in this study is also beneficial for a variety of biophysical and spectroscopic techniques, including solution NMR and magic-angle-spinning (MAS) NMR for a wide range of temperatures.
- Published
- 2015
44. Characterization and Calculation of a Cytochrome c−Cytochrome b5 Complex Using NMR Data
- Author
-
Sang Choul Im, Erik R. P. Zuiderweg, Shashank Deep, and Lucy Waskell
- Subjects
Surface Properties ,Analytical chemistry ,environment and public health ,Biochemistry ,Article ,chemistry.chemical_compound ,Multienzyme Complexes ,Amide ,Protein Interaction Mapping ,Cytochrome b5 ,Escherichia coli ,Side chain ,Animals ,Horses ,Binding site ,Nuclear Magnetic Resonance, Biomolecular ,Heme ,Carbon Isotopes ,Nitrogen Isotopes ,biology ,Chemistry ,Cytochrome c ,Titrimetry ,Cytochromes c ,Recombinant Proteins ,enzymes and coenzymes (carbohydrates) ,Crystallography ,Cytochromes b5 ,Docking (molecular) ,embryonic structures ,cardiovascular system ,biology.protein ,Cattle ,Titration ,Protein Binding - Abstract
To identify the binding site for bovine cytochrome b(5) (cyt b(5)) on horse cytochrome c (cyt c), cross-saturation transfer NMR experiments were performed with (2)H- and (15)N-enriched cyt c and unlabeled cyt b(5). In addition, chemical shift changes of the cyt c backbone amide and side chain methyl resonances were monitored as a function of cyt b(5) concentration. The chemical shift changes indicate that the complex is in fast exchange, and are consistent with a 1:1 stoichiometry. A K(a) of (4 +/- 3) x 10(5) M(-)(1) was obtained with a lower limit of 855 s(-)(1) for the dissociation rate of the complex. Mapping of the chemical shift variations and intensity changes upon cross-saturation NMR experiments in the complex reveals a single, contiguous interaction interface on cyt c. Using NMR data as constraints, a protein docking program was used to calculate two low-energy model complex clusters. Independent calculations of the effect of the cyt b(5) heme ring current-induced magnetic dipole on cyt c were used to discriminate between the different models. The interaction surface of horse cyt c in the current experimentally constrained model of the cyt c-cyt b(5) complex is similar but not identical to the interface predicted in yeast cyt c by Brownian dynamics and docking calculations. The occurrence of different amino acids at the protein-protein interface and the dissimilar assumptions employed in the calculations can largely account for the nonidentical interfaces.
- Published
- 2005
45. Mapping the Binding Interface of the Cytochrome b5−Cytochrome c Complex by Nuclear Magnetic Resonance
- Author
-
Erik R. P. Zuiderweg, Weiping Shao, Sang Choul Im, and Lucy Waskell
- Subjects
Models, Molecular ,Protein Conformation ,Surface Properties ,Peptide Mapping ,environment and public health ,Biochemistry ,chemistry.chemical_compound ,Nuclear magnetic resonance ,Cytochrome b5 ,Side chain ,Animals ,Horses ,Binding site ,Nuclear Magnetic Resonance, Biomolecular ,Heme ,Conserved Sequence ,Carbon Isotopes ,Binding Sites ,Nitrogen Isotopes ,biology ,Cytochrome b ,Cytochrome c ,Chemical shift ,Cytochromes c ,Nuclear magnetic resonance spectroscopy ,enzymes and coenzymes (carbohydrates) ,Cytochromes b5 ,Models, Chemical ,chemistry ,embryonic structures ,cardiovascular system ,biology.protein ,Thermodynamics ,Cattle ,Protein Binding - Abstract
The interaction between bovine cytochrome b(5) (cyt b(5)) and horse heart cytochrome c (cyt c) is investigated by NMR spectroscopy. Chemical shifts of cyt b(5) backbone resonances and side chain methyl resonances were monitored as a function of cyt c concentration. The shifts are small but saturatable and indicate that the binding of cyt b(5) with cyt c is in fast exchange. An equilibrium association constant of (6 +/- 3) x 10(4) M(-1) was obtained with a lower limit of 180 s(-1) for the dissociation rate of the complex. To resolve considerable ambiguities in the interpretation of the chemical shift mapping, (15)N relaxation experiments and cross-saturation experiments were used as alternative methods to map the cyt b(5)-cyt c binding interface. Results from the three experiments combined demonstrate that the conserved negatively charged region of cyt b(5) surrounding the solvent-exposed heme edge is involved in the interaction with cyt c. These data support the models proposed by Salemme and Mauk [(1976) J. Mol. Biol. 102, 563-568; (1993) Biochemistry 32, 6613-6623].
- Published
- 2003
46. Preparation and Characterization of a 5‘-DeazaFAD T491V NADPH−Cytochrome P450 Reductase
- Author
-
A. Sami Saribas, Anna L. Shen, Larry Gruenke, Sang Choul Im, Lucy Waskell, Haoming Zhang, and Charles B. Kasper
- Subjects
Flavin Mononucleotide ,Stereochemistry ,Flavoprotein ,Flavin group ,Reductase ,Dithionite ,Biochemistry ,Redox ,Cofactor ,chemistry.chemical_compound ,Escherichia coli ,Animals ,NADPH-Ferrihemoprotein Reductase ,Binding Sites ,biology ,Hydroquinone ,Chemistry ,Cytochrome c ,Protein Structure, Tertiary ,Rats ,Kinetics ,Amino Acid Substitution ,Flavin-Adenine Dinucleotide ,biology.protein ,Spectrophotometry, Ultraviolet ,Oxidation-Reduction - Abstract
NADPH-cytochrome P450 reductase is a flavoprotein which contains both an FAD and FMN cofactor. Since the distribution of electrons is governed solely by the redox potentials of the cofactors, there are nine different waysthe electrons can be distributed and hence nine possible unique forms of the protein. More than one species of reductase will exist at a given level of oxidation except when the protein is either totally reduced or oxidized. In an attempt to unambiguously characterize the redox properties of the physiologically relevant FMNH2 form of the reductase, the T491V mutant of NADPH-cytochrome P450 reductase has been reconstituted with 5'-deazaFAD which binds to the FAD-binding site of the reductase with a Kd of 94 nM. The 5'-deazaFAD cofactor does not undergo oxidation or reduction under our experimental conditions. The molar ratio of FMN to 5'-deazaFAD in the reconstituted reductase was 1.1. Residual FAD accounted for less than 5% of the total flavins. Addition of 2 electron equivalents to the 5'-deazaFAD T491V reductase from dithionite generated a stoichiometric amount of the FMN hydroquinone form of the protein. The 5'-deazaFAD moiety remained oxidized under these conditions due to its low redox potential (-650 mV). The 2-electron-reduced 5'-deazaFAD reductase was capable of transferring only a single electron from its FMN domain to its redox partners, ferric cytochrome c and cytochrome b5. Reduction of the cytochromes and oxidation of the reductase occurred simultaneously. The FMNH2 in the 5'-deazaFAD reductase autoxidizes with a first-order rate constant of 0.007 s-1. Availability of a stable NADPH-cytochrome P450 reductase capable of donating only a single electron to its redox partners provides a unique tool for investigating the electron-transfer properties of an intact reductase molecule.
- Published
- 2003
47. Effects of membrane mimetics on cytochrome P450-cytochrome b5 interactions characterized by NMR spectroscopy
- Author
-
Ayyalusamy Ramamoorthy, Lucy Waskell, Meng Zhang, Rui Huang, and Sang Choul Im
- Subjects
Stereochemistry ,Model lipid bilayer ,Biochemistry ,digestive system ,Cell membrane ,Electron Transport ,Biomimetic Materials ,Multienzyme Complexes ,Cytochrome b5 ,medicine ,polycyclic compounds ,Animals ,Lipid bilayer ,Cytochrome P450 Family 2 ,Protein Structure, Quaternary ,Molecular Biology ,Nuclear Magnetic Resonance, Biomolecular ,Chemistry ,Peripheral membrane protein ,Cell Membrane ,Cytochrome P450 reductase ,Membrane Proteins ,Cell Biology ,Electron transport chain ,Protein Structure, Tertiary ,medicine.anatomical_structure ,Cytochromes b5 ,Membrane protein ,lipids (amino acids, peptides, and proteins) ,Aryl Hydrocarbon Hydroxylases ,Rabbits ,Molecular Biophysics ,Protein Binding - Abstract
Mammalian cytochrome P450 (P450) is a membrane-bound monooxygenase whose catalytic activities require two electrons to be sequentially delivered from its redox partners: cytochrome b5 (cytb5) and cytochrome P450 reductase, both of which are membrane proteins. Although P450 functional activities are known to be affected by lipids, experimental evidence to reveal the effect of membrane on P450-cytb5 interactions is still lacking. Here, we present evidence for the influence of phospholipid bilayers on complex formation between rabbit P450 2B4 (CYP2B4) and rabbit cytb5 at the atomic level, utilizing NMR techniques. General line broadening and modest chemical shift perturbations of cytb5 resonances characterize CYP2B4-cytb5 interactions on the intermediate time scale. More significant intensity attenuation and a more specific protein-protein binding interface are observed in bicelles as compared with lipid-free solution, highlighting the importance of the lipid bilayer in stabilizing stronger and more specific interactions between CYP2B4 and cytb5, which may lead to a more efficient electron transfer. Similar results observed for the interactions between CYP2B4 lacking the transmembrane domain (tr-CYP2B4) and cytb5 imply interactions between tr-CYP2B4 and the membrane surface, which might assist in CYP2B4-cytb5 complex formation by orienting tr-CYP2B4 for efficient contact with cytb5. Furthermore, the observation of weak and nonspecific interactions between CYP2B4 and cytb5 in micelles suggests that lipid bilayer structures and low curvature membrane surface are preferable for CYP2B4-cytb5 complex formation. Results presented in this study provide structural insights into the mechanism behind the important role that the lipid bilayer plays in the interactions between P450s and their redox partners.
- Published
- 2014
48. Probing the Transmembrane Structure and Dynamics of Microsomal NADPH-cytochrome P450 oxidoreductase by Solid-State NMR
- Author
-
Nataliya Popovych, Ivan Hung, Rui Huang, Ayyalusamy Ramamoorthy, Meng Zhang, Zhehong Gan, Kazutoshi Yamamoto, Sang Choul Im, and Lucy Waskell
- Subjects
Models, Molecular ,Magnetic Resonance Spectroscopy ,Flavin Mononucleotide ,Stereochemistry ,Biophysics ,Flavin mononucleotide ,Biology ,Protein Structure, Secondary ,Cell membrane ,chemistry.chemical_compound ,FMN binding ,Microsomes ,medicine ,Animals ,Lipid bilayer ,NADPH-Ferrihemoprotein Reductase ,Membranes ,New and Notable ,Endoplasmic reticulum ,Cell Membrane ,Membrane Proteins ,Transmembrane protein ,Protein Structure, Tertiary ,Rats ,Transmembrane domain ,medicine.anatomical_structure ,Membrane protein ,chemistry - Abstract
NADPH-cytochrome P450 oxidoreductase (CYPOR) is an essential redox partner of the cytochrome P450 (cyt P450) superfamily of metabolic enzymes. In the endoplasmic reticulum of liver cells, such enzymes metabolize ∼75% of the pharmaceuticals in use today. It is known that the transmembrane domain of CYPOR plays a crucial role in aiding the formation of a complex between CYPOR and cyt P450. Here we present the transmembrane structure, topology, and dynamics of the FMN binding domain of CYPOR in a native membrane-like environment. Our solid-state NMR results reveal that the N-terminal transmembrane domain of CYPOR adopts an α-helical conformation in the lipid membrane environment. Most notably, we also show that the transmembrane helix is tilted ∼13° from the lipid bilayer normal, and exhibits motions on a submillisecond timescale including rotational diffusion of the whole helix and fluctuation of the helical director axis. The approaches and the information reported in this study would enable further investigations on the structure and dynamics of the full-length NADPH-cytochrome P450 oxidoreductase and its interaction with other membrane proteins in a membrane environment.
- Published
- 2014
49. Pick a ska or reggae beat? Rates of conformational switching in multi‐domain flavoproteins and their relationship to electron flux through the enzymes (768.8)
- Author
-
May Pearl, Mekki Bayachou, Dennis J. Stuehr, Sang Choul Im, Lucy Waskell, and Mohammad Mahfuzul Haque
- Subjects
chemistry.chemical_classification ,biology ,Flavoprotein ,Beat (acoustics) ,Biochemistry ,Multi domain ,Enzyme ,Nuclear magnetic resonance ,chemistry ,Chemical physics ,Electron flux ,Genetics ,biology.protein ,Molecular Biology ,Biotechnology - Published
- 2014
50. Cellular solid-state NMR investigation of a membrane protein using dynamic nuclear polarization
- Author
-
Lucy Waskell, Kazutoshi Yamamoto, Marc A. Caporini, Sang Choul Im, and Ayyalusamy Ramamoorthy
- Subjects
Magnetic Resonance Spectroscopy ,Lipid bilayers ,Molecular Sequence Data ,Biophysics ,In-cell NMR spectroscopy ,010402 general chemistry ,01 natural sciences ,Biochemistry ,Article ,03 medical and health sciences ,Electron transfer ,Dynamic nuclear polarization ,Magic angle spinning ,Escherichia coli ,Solid-state NMR spectroscopy ,Amino Acid Sequence ,Lipid bilayer ,030304 developmental biology ,0303 health sciences ,Chemistry ,Membrane Proteins ,Cell Biology ,Nuclear magnetic resonance spectroscopy ,Polarization (waves) ,0104 chemical sciences ,Cytochromes b5 ,Solid-state nuclear magnetic resonance ,Membrane protein ,Structural biology - Abstract
While an increasing number of structural biology studies successfully demonstrate the power of high-resolution structures and dynamics of membrane proteins in fully understanding their function, there is considerable interest in developing NMR approaches to obtain such information in a cellular setting. As long as the proteins inside the living cell tumble rapidly in the NMR timescale, recently developed in-cell solution NMR approaches can provide 3D structural information. However, there are numerous challenges to study membrane proteins inside a cell. Research in our laboratory is focused on developing a combination of solid-state NMR and biological approaches to overcome these challenges in order to obtain high-resolution structural insights into electron transfer processes mediated by membrane-bound proteins like mammalian cytochrome-b5, cytochrome-P450 and cytochrome-P450-reductase. In this study, we demonstrate the feasibility of using dynamic nuclear polarization (DNP) magic angle spinning (MAS) NMR spectroscopy for in-cell studies on a membrane-anchored protein. Our experimental results obtained from 13C-labeled membrane-anchored cytochrome-b5 in native Escherichia coli cells show a ~16-fold DNP signal enhancement. Further, results obtained from a 2D 13C/13C chemical shift correlation MAS experiment demonstrate the feasibility of suppressing the background signals from other cellular contents for high-resolution structural studies on membrane proteins. We believe that this study would pave new avenues for high-resolution structural studies on a variety of membrane-associated proteins and their complexes in the cellular context to fully understand their functional roles in physiological processes. This article is part of a Special Issue entitled: NMR Spectroscopy for Atomistic Views of Biomembranes and Cell Surfaces. Guest Editors: Lynette Cegelski and David P. Weliky.
- Published
- 2014
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