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

Numerous investigations have focused on DNA damage induced by ionizing radiation; however, photoionization threshold energies of nucleic acid components in aqueous solution are not known. Herein, data from gas-phase photoelectron experiments have been combined with results from selfconsistent field and post-self-consistent field molecular orbital calculations and with theoretical Gibbs free energies of hydration to describe aqueous ionization energies of 2-deoxythymidine 5-phosphate (5-dTMP ؊ ) and 2-deoxycytidine 5-phosphate (5-dCMP ؊ ). For the test molecules, indole and tryptophan, this approach yields aqueous ionization energies (4.46 and 4.58 eV, respectively) in agreement with experimental values (4.35 and 4.45 eV). When uridine and 2-deoxythymidine ionization energies are evaluated, the results agree with recent data from 193-nm laser measurements indicating that uridine ionization occurs via a one-photon event. For 5-dCMP ؊ and 5-dTMP ؊ , a comparison of aqueous ionization energies with gas-phase ionization potentials (IPs) indicates that hydration alters the relative energies of ionization events. In the gas phase, phosphate vertical IPs are ϳ1.3 eV smaller than base IPs. In aqueous solution, the base and phosphate ionization energies are more similar, and only differ by ϳ0.5 eV. For 5-dCMP ؊ and 5-dTMP ؊ , the increased favorableness of base ionization, which accompanies hydration, is consistent with experimental data indicating that, at 77 K in aqueous perchlorate glasses, the primary photoionization pathway involves base ionization followed by deprotonation.Nucleotide ionization plays an important role in mechanisms resulting in DNA damage caused by high-energy photons (1-4). However, there is little information about threshold energies required for DNA ionization in aqueous solution, except that they lie in the range of 4 to 7 eV (5, 6). In earlier work, vertical and adiabatic ionization potentials (IPs) were provided for gas-phase nucleotide components and model compounds (7-10). More recently, experimental data on nucleotide component model compounds has been combined with theoretical results to evaluate IPs for intact gas-phase nucleotides (11)(12)(13)(14). Herein, gasphase UV photoelectron data were used with results from molecular orbital calculations at the self-consistent field (SCF) and post-SCF levels to provide valence electron IPs of the gas-phase anionic nucleotides 2Ј-deoxycytidine 5Ј-phosphate (5Ј-dCMP Ϫ ) (12, 15), 2Ј-deoxythymidine 5Ј-phosphate (5Ј-dTMP Ϫ ) (15), 2Ј-deoxyguanosine 5Ј-phosphate (5Ј-dGMP Ϫ ) (11,13,15,16), and 2Ј-deoxyadenosine 5Ј-phosphate (5Ј-dAMP Ϫ ) (14, 15). In all cases, the lowest IP is associated with the negatively charged phosphate group. However, these earlier gas-phase results do not provide information about nucleotide ionization under physiological conditions.Water and counterion interactions are among the most important that DNA encounters in biological environments, and the effects of these interactions on nucleotide IPs were examined in gas-pha...

To explore the mechanism of homotropic cooperativity in human cytochrome P450 3A4 (CYP3A4) we studied the interactions of the enzyme with 1-pyrenebutanol (1-PB), 1-pyrenemethylamine (PMA), and bromocriptine by FRET from the substrate fluorophore to the heme, and by absorbance spectroscopy. These approaches combined with an innovative setup of titration-by-dilution and continous variation (Job's titration) experiments allowed us to probe the relationship between substrate binding and the subsequent spin transition caused by 1-PB or bromocriptine or the Type-II spectral changes caused by PMA. The 1-PB-induced spin shift in CYP3A4 reveals prominent homotropic cooperativity, which is characterized by a Hill coefficient of 1.8 ± 0.3 (S 50 = 8.0 ± 1.1 µM). In contrast, the interactions of CYP3A4 with bromocriptine or PMA reveal no cooperativity, exhibiting K D values of 0.31 ± 0.08 µM and 6.7 ± 1.9 µM, respectively. The binding of all three substrates monitored by FRET in titration-by-dilution experiments at an enzyme:substrate ratio of 1 reveals a simple bimolecular interaction with K D values of 0.16 ± 0.09, 4.8 ± 1.4, and 0.18 ± 0.09 µM for 1-PB, PMA, and bromocriptine respectively. Correspondingly, the Job's titration experiments showed that the 1-PB-induced spin shift reflects the formation of a complex of the enzyme with two substrate molecules, while bromocriptine and PMA exhibit 1:1 binding stoichiometry. Combining the results of Job's titrations with the value of K D obtained in our FRET experiments, we demonstrate that the interactions of CYP3A4 with 1-PB obey a sequential binding mechanism, where the spin transition is triggered by the binding of 1-PB to the low-affinity site, which becomes possible only upon saturation of the high-affinity site.Keywords substrate binding; cooperativity; conformers; spin equilibrium Recent studies of function and regulation of cytochromes P450 reveal increasing attention to the mechanisms and pharmacological significance of homo-and heterotropic cooperativity observed in various mammalian P450 species (1-8). The most prominent examples of these phenomena are observed with cytochrome P450 3A4 (CYP3A4), the most abundant P450 in human liver. CYP3A4 is responsible for the metabolism of a broad range of drug substrates, and studies of cooperativity of this enzyme are of vital importance to our efforts to elucidate the mechanisms of drug metabolism in humans (9). However, despite extensive studies, a general mechanism of cooperativity remains obscure. † This research was supported by NIH grants GM54995 (JRH), and Center grant ES06676 (JRH). NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author ManuscriptThe prevailing hypothesis is that cytochromes P450 exhibiting cooperativity accommodate multiple substrate molecules in one large binding pocket (10)(11)(12). A loose fit of a single substrate molecule requires the binding of a second ligand for efficient binding and/or catalysis (11-13). However, the possibility that P450 cooperativity reflects a true case of a...

Background: Cytochrome P450 3A4 (CYP3A4) can bind several substrate molecules simultaneously and exhibits cooperativity. Results: Ligand binding in the active site is preceded by functionally important interactions at a distinct peripheral site. Conclusion: The mechanism of cooperativity involves a ligand-induced allosteric transition. Significance: Allosteric mechanism suggested by our results transforms the view of the grounds and significance of CYP3A4 cooperativity.

Chemokines play a fundamental role in trafficking of immune cells and in host defense against infection. The role of chemokines in the recruitment process is highly regulated spatially and temporally and involves interactions with G protein-coupled receptors and cell surface glycosaminoglycans. The dynamic equilibrium between chemokine monomers and dimers, both free in solution and in cell surface-bound forms, regulates different components of recruitment such as chemotaxis and receptor signaling. The binding and activity of the chemokine interleukin-8 (IL-8) for its receptors, previously studied using "trapped" non-associating monomers and non-dissociating dimers, show that the monomer has a native-like function but support conflicting roles for the dimer. We have measured the binding of native IL-8 to the CXCR1 N-domain, using isothermal titration calorimetry and sedimentation equilibrium techniques. The N-domain constitutes a critical binding site, and IL-8 binding affinity to the receptor N-domain is in the same concentration range as the IL-8 monomerdimer equilibrium. We observed that only the IL-8 monomer, and not the dimer, is competent in binding the receptor N-domain. Based on our results, we propose that IL-8 dimerization functions as a negative regulator for the receptor function and as a positive regulator for binding to glycosaminoglycans and that both play a role in the neutrophil recruitment process.

Background Hepatic steatosis (fatty liver), an early and reversible stage of alcoholic liver disease, is characterized by triglyceride deposition in hepatocytes, that can advance to steatohepatitis, fibrosis, cirrhosis, and ultimately to hepatocellular carcinoma. In the present work, we studied altered plasma and hepatic lipid metabolome (lipidome) to understand the mechanisms and lipid pattern of early stage alcohol induced-fatty liver. Methods Male Fischer 344 rats were fed 5% alcohol in a Lieber-DeCarli diet. Control rats were pair-fed an equivalent amount of maltose-dextrin. After one month, animals were sacrificed and plasma collected. Livers were excised for morphological, immunohistochemical, and biochemical studies. The lipids from plasma and livers were extracted with methyl-tert-butyl ether and analyzed by 750/800 MHz proton nuclear magnetic resonance (1H NMR) and phosphorus (31P) NMR spectroscopy on a 600 MHz spectrometer. The NMR data were then subjected to multivariate statistical analysis. Results Hemotoxylin & Eosin and Oil Red O stained liver sections showed significant fatty infiltration. Immunohistochemical analysis of liver sections from ethanol-fed rats showed no inflammation (absence of CD3 positive cells) or oxidative stress (absence of malondialdehyde reactivity or 4-hydroxynonenal positive straining). Cluster analysis and principal component analysis of 1H NMR data of lipid extracts of both plasma and livers showed a significant difference in the lipid metabolome of ethanol-fed vs. control rats. 31P NMR data of liver lipid extracts showed significant changes in phospholipids similar to 1H NMR data. 1H NMR data of plasma and liver reflected several changes while comparison of 1H NMR and 31P NMR data offered a correlation among the phospholipids. Conclusions Our results show that alcohol consumption alters metabolism of cholesterol, triglycerides and phospholipids that could contribute to the development of fatty liver. These studies also indicate that fatty liver precedes oxidative stress and inflammation. The similarities observed in plasma and liver lipid profiles offer a potential methodology for detecting early stage alcohol-induced fatty liver disease by analyzing the plasma lipid profile.

Interleukin-8 (IL-8), a member of the chemokine superfamily, exists as both monomers and dimers, and mediates its function by binding to neutrophil CXCR1 and CXCR2 receptors that belong to the G protein-coupled receptor class. It is now well established that the monomer functions as a high-affinity ligand, but the binding affinity of the dimer remains controversial. The approximately 1000-fold difference between monomer-dimer equilibrium constant (microM) and receptor binding constant (nM) of IL-8 does not allow receptor-binding affinity measurements of the native IL-8 dimer. In this study, we overcame this roadblock by creating a "trapped" nondissociating dimer that contains a disulfide bond across the dimer interface at the 2-fold symmetry point. The NMR studies show that the structure of this trapped dimer is indistinguishable from the native dimer. The trapped dimer, compared to a trapped monomer, bound CXCR1 with approximately 70-fold and CXCR2 with approximately 20-fold lower affinities. Receptor binding involves two interactions, between the IL-8 N-loop and receptor N-domain residues, and between IL-8 N-terminal and receptor extracellular loop residues. In contrast to a trapped monomer that bound an isolated CXCR1 N-domain peptide with microM affinity, the trapped dimer failed to show any binding, indicating that dimerization predominantly perturbs the binding of only the N-loop residues. These results demonstrate that only the monomer is a high-affinity ligand for both receptors, and also provide a structural basis for the lower binding affinity of the dimer.

Alcoholic liver disease (ALD) is a serious health problem with significant morbidity and mortality. In this study we examined the progression of ALD along with lipidomic changes in rats fed ethanol for 2 and 3 months to understand the mechanism, and identify possible biomarkers. Male Fischer 344 rats were fed 5% ethanol or caloric equivalent of maltose-dextrin in a Lieber-DeCarli diet. Animals were killed at the end of 2 and 3 months and plasma and livers were collected. Portions of the liver were fixed for histological and immunohistological studies. Plasma and the liver lipids were extracted and analyzed by nuclear magnetic resonance (NMR) spectroscopy. A time dependent fatty infiltration was observed in the livers of ethanol-fed rats. Mild inflammation and oxidative stress were observed in some ethanol-fed rats at 3 months. The multivariate and principal component analysis of proton and phosphorus NMR spectroscopy data of extracted lipid from the plasma and livers showed segregation of ethanol-fed groups from the pair-fed controls. Significant hepatic lipids that were increased by ethanol exposure included fatty acids and triglycerides, whereas phosphatidylcholine (PC) decreased. However, both free fatty acids and PC decreased in the plamsa. In liver lipids unsaturation of fatty acyl chains increased, contrary to plasma, where it decreased. Our studies confirm that over-accumulation of lipids in ethanol-induced liver steatosis accompanied by mild inflammation on long duration of ethanol exposure. Identified metabolic profile using NMR lipidomics could be further explored to establish biomarker signatures representing the etiopathogenesis, progression and/or severity of ALD.