Biomedical subjects

S G Sligar

Publications and source records attributed to S G Sligar.

At least 19 recordsLinked to original sources

PubMed2004

Directed self-assembly of monodisperse phospholipid bilayer Nanodiscs with controlled size.

Using a recently described self-assembly process (Bayburt, T. H.; Grinkova, Y. V.; Sligar, S. G. Nano Letters 2002, 2, 853-856), we prepared soluble monodisperse discoidal lipid/protein particles with controlled size and composition, termed Nanodiscs, in which the fragment of dipalmitoylphosphatidylcholine (DPPC) bilayer is surrounded by a helical protein belt. We have customized the size of these particles by changing the length of the amphipathic helical part of this belt, termed membrane scaffold protein (MSP). Herein we describe the design of extended and truncated MSPs, the optimization of self-assembly for each of these proteins, and the structure and composition of the resulting Nanodiscs. We show that the length of the protein helix surrounding the lipid part of a Nanodisc determines the particle diameter, as measured by HPLC and small-angle X-ray scattering (SAXS). Using different scaffold proteins, we obtained Nanodiscs with the average size from 9.5 to 12.8 nm with a very narrow size distribution (+/-3%). Functionalization of the N-terminus of the scaffold protein does not perturb their ability to form homogeneous discoidal structures. Detailed analysis of the solution scattering confirms the presence of a lipid bilayer of 5.5 nm thickness in Nanodiscs of different sizes. The results of this study provide an important structural characterization of self-assembled phospholipid bilayers and establish a framework for the design of soluble amphiphilic nanoparticles of controlled size.

1,2-Dipalmitoylphosphatidylcholine↗

PubMed2003

Haem-oxygen reactive intermediates: catalysis by the two-step.

The catalytic schemes of a variety of haem enzymes, including the P450 mono-oxygenases, consist of a number of common reactive haem-oxygen adducts. The characterization of these intermediates by optical and EPR spectroscopies has reinforced the similarity of these intermediate states in a number of haem enzyme systems. Furthermore, the reactivity of these states in P450 and horseradish peroxidase, in which multiple potent oxidants are formed, provides a paradigm for many other haem enzymes.

Catalysis↗

PubMed2002

Experimental and theoretical high pressure strategies for investigating protein-nucleic acid assemblies.

A method was developed to investigate the stability of protein-nucleic acid complexes using hydrostatic pressure during electrophoretic gel mobility shift analysis. The initial system probed by this technique was the well-characterized cognate BamHI-DNA complex. Band shift analysis at several elevated pressures found the equilibrium dissociation (K(d)) constant to be dependent on pressure, which allowed the volume change of dissociation (deltaV) to be calculated. In order to describe the effects of pressure on the specific BamHI-DNA complex at the molecular level, molecular dynamics simulations at both ambient and elevated pressure was performed. Comparison of the simulation trajectories identified several individual BamHI-DNA contacts that are disrupted due to pressure. The disruption of these contacts can be attributed to an observed pressure-induced increase in hydration at the protein-DNA interface during the elevated pressure simulation.

Computer Simulation↗

PubMed2001

Characterization of the oxygenated intermediate of the thermophilic cytochrome P450 CYP119.

Using UV-Vis, resonance Raman, and EPR spectroscopy we have studied the properties of the oxygenated ferrous cytochrome P450 from Sulfolobus solfataricus, (CYP119). The recently determined crystal structure of CYP119 is compared with other available structures of P450s, and detailed structural and spectroscopic analyses are reported. With several structural similarities to CYP102, such as in-plane iron position and a shorter iron-proximal ligand bond, CYP119 shows low-spin conformation preference in the ferric form and partially in the ferrous form at low temperatures. These structural features can explain the fast autoxidation of the oxyferrous complex of CYP119. Finally, we report the first UV-Vis and EPR spectra of the cryoradiolytically reduced oxygenated intermediate of CYP119. The primary reduced intermediate, a hydroperoxo-ferric complex of CYP119, undergoes a 'peroxide shunt' pathway during gradual annealing at 170-195 K and returns to the low-spin ferric form.

Archaeal Proteins↗

PubMed2001

Revelation of ternary complexes between redox partners in cytochrome P450-containing monooxygenase systems by the optical biosensor method.

Formation of binary and ternary complexes in the water-soluble cytochrome P450cam (P450cam)-containing as well as in the membrane P4502B4(2B4)- and the mixed P450scc-containing monooxygenase systems was investigated in real time by the 'resonant mirror' optical biosensor method. It was shown that the inter-protein electron transfer occurs not only during complex formation but also upon random collision--as was the case with the d-Fp/d-b5 pair (2B4 system). Binary complexes may be either facilitative to electron transfer (electron-transfer complexes) or prohibitive to it (non-productive complexes). Although the binary PdR/Pd and P450cam/Pd complex formation (within the P450cam-system) as well as the binary AdR/Ad and P450scc/Ad complex formation (within the P450scc-system) does occur, the lifetimes of these complexes formed are several orders of magnitude higher than the time required for realization of a complete hydroxylation cycle. At the same time, the lifetimes of the ternary PdR/Pd/P450cam and AdR/Ad/P450scc complexes are sufficient to permit the realization of a complete hydroxylation cycle in either of these systems. For the membrane P450 2B4 system, the formation of both the binary (Fp/2B4 and 2B4/b5) and ternary (Fp/2B4/b5) complexes was registered. The lifetimes of the binary Fp/2B4 and the ternary Fp/2B4/b5 complexes are sufficient for realization of a complete hydroxylation cycle in each of them.

Biosensing Techniques↗

PubMed2001

Molecular recognition in the p450cam monooxygenase system: direct monitoring of protein-protein interactions by using optical biosensor.

A real-time optical biosensor study on the interactions between putidaredoxin reductase (PdR), putidaredoxin (Pd), and cytochrome P450cam (P450cam) within the P450cam system was conducted. The binary Pd/P450cam and Pd/PdR complexes were revealed and kinetically characterized. The dominant role of electrostatic interactions in formation of productive electron transfer complexes was demonstrated. It was found that Pd/P450cam complex formation and decay obeys biphasic kinetics in contrast to the monophasic one for complexes formed by other redox partners within the system. Evidence for PdR/P450cam complex formation was obtained. It was found that, in contrast to Pd, which binds only to its redox partners, PdR and P450cam were able to form PdR/PdR and P450cam/P450cam complexes. A ternary PdR/Pd/P450cam complex was also registered. Its lifetime was sufficient to permit up to 60 turnovers to occur. The binding of Pd to P450cam and to PdR within the ternary complex occurred at distinct sites, with Pd serving as a bridge between the two proteins.

Binding Sites↗

PubMed2001

Resonance Raman and EPR investigations of the D251N oxycytochrome P450cam/putidaredoxin complex.

We have performed resonance Raman and electron paramagnetic resonance (EPR) studies on the dioxygen bound state of the D251N mutant of cytochrome P450cam (oxy-P450cam) and its complex with reduced putidaredoxin (Pd). The D251N oxy-P450cam/Pd complex has a perturbed proton delivery mechanism and shows a significantly red-shifted UV-visible spectrum as observed in Benson et al. [Benson, D. E., Suslick, K. S., and Sligar, S. G. (1997) Biochemistry 36, 5104-5107]. The red shift has been interpreted to indicate a major perturbation of the electronic structure of the oxy-heme complex. However, we find no evidence that electron transfer has occurred from Pd to the heme active site of D251N oxy-P450cam. This suggests that both electron and proton transfer are perturbed by the D251N mutation and that these processes may be coupled. Three oxygen isotope sensitive Raman features are identified in the Pd complex, and occur at 1137, 536, and 399 cm(-1). These values are not significantly different from those for WT or D251N oxy-P450cam. However, a careful examination of the oxygen stretching feature near 1137 cm(-1) reveals the presence of three peaks at 1131, 1138, and 1146 cm(-1), which we attribute to the presence of conformational substates in oxy-P450cam. A significant change in the conformational substate population is observed for the D251N oxy-P450cam when the Pd complex is formed. We suggest that the conformational population redistribution of oxy-P450cam, along with the red-shifted electronic spectra, reflects a structural equilibrium of the oxy-heme that is perturbed upon Pd binding. We propose that this structural perturbation is connected to the effector function of Pd and may involve changes in the electron donation properties of the thiolate ligand.

Amino Acid Substitution↗

PubMed2001

Hydroxylation of camphor by reduced oxy-cytochrome P450cam: mechanistic implications of EPR and ENDOR studies of catalytic intermediates in native and mutant enzymes.

We have employed gamma-irradiation at cryogenic temperatures (77 K and also approximately 6 K) of the ternary complexes of camphor, dioxygen, and ferro-cytochrome P450cam to inject the "second" electron of the catalytic process. We have used EPR and ENDOR spectroscopies to characterize the primary product of reduction as well as subsequent states created by annealing reduced oxyP450, both the WT enzyme and the D251N and T252A mutants, at progressively higher temperatures. (i) The primary product upon reduction of oxyP450 4 is the end-on, "H-bonded peroxo" intermediate 5A. (ii) This converts even at cryogenic temperatures to the hydroperoxo-ferriheme species, 5B, in a step that is sensitive to these mutations. Yields of 5B are as high as 40%. (iii) In WT and D251N P450s, brief annealing in a narrow temperature range around 200 K causes 5B to convert to a product state, 7A, in which the product 5-exo-hydroxycamphor is coordinated to the ferriheme in a nonequilibrium configuration. Chemical and EPR quantitations indicate the reaction pathway involving 5B yields 5-exo-hydroxycamphor quantitatively. Analogous (but less extensive) results are seen for the alternate substrate, adamantane. (iv) Although the T252A mutation does not interfere with the formation of 5B, the cryoreduced oxyT252A does not yield product, which suggests that 5B is a key intermediate at or near the branch-point that leads either to product formation or to nonproductive "uncoupling" and H(2)O(2) production. The D251N mutation appears to perturb multiple stages in the catalytic cycle. (v) There is no spectroscopic evidence for the buildup of a high-valence oxyferryl/porphyrin pi-cation radical intermediate, 6. However, ENDOR spectroscopy of 7A in H(2)O and D(2)O buffers shows that 7A contains hydroxycamphor, rather than water, bound to Fe(3+), and that the proton removed from the C(5) carbon of substrate during hydroxylation is trapped as the hydroxyl proton. This demonstrates that hydroxylation of substrates by P450cam in fact occurs by the formation and reaction of 6. (vi) Annealing at > or = 220 K converts the initial product state 7A to the equilibrium product state 7, with the transition occurring via a second nonequilibrium product state, 7B, in the D251N mutant; in states 7B and 7 the hydroxycamphor hydroxyl proton no longer is trapped. (vii) The present results are discussed in the context of other efforts to detect intermediates in the P450 catalytic cycle.

Camphor↗

PubMed2001

Hydrogen-bonding interactions in the active sites of cytochrome P450cam and its site-directed mutants.

Resonance Raman spectroscopy is applied to the cyanide adducts of cytochrome P450cam and its T252A and D251N site-directed mutants, both in their substrate-free and camphor-bound forms, to probe active-site heme structure and, in particular, interactions of the FeCN fragment with potential active-site H-bond donors. In contrast to the ferrous CO and ferric NO adducts, which form only essentially linear (slightly distorted) FeXY fragments, the spectra of the ferric CN(-) adducts provide clear evidence the for the existence of an additional, rather highly bent, conformer; that is, the cyanide complexes form both linear and bent conformers in both the substrate-free and substrate-bound forms. Formation of this bent conformer is most reasonably attributed to the presence of off-axis H-bond donors, which induce distortion on the FeCN fragment but not the FeCO and FeNO fragments, which are poorer H-bond acceptors. For all three proteins, the substrate-free form exhibits a complex spectral pattern which arises because one of the modes associated with the FeCN fragment is coupled with two heme macrocycle deformation modes. Significantly, no evidence for such coupling is observed in the spectra of the camphor-bound forms. While various unknown factors may possibly give rise to selective activation of such coupling in the substrate-free derivative, given the known facts about the active-site architecture of this enzyme, a plausible explanation is that the bent conformer is oriented toward the water-filled substrate-binding site in the substrate-free form, but oppositely, toward the proposed proton delivery shuttle, in the substrate-bound form. Sensitivity of the FeCN modes to H(2)O/D(2)O exchange in the two camphor-bound mutants, which is apparently absent for the camphor-bound native protein, is most reasonably attributed to the known presence of extra water in the active sites of these mutants.

Bacterial Proteins↗

PubMed2001

Cryotrapped reaction intermediates of cytochrome p450 studied by radiolytic reduction with phosphorus-32.

Unstable reaction intermediates of the cytochrome P450 catalytic cycle have been prepared at cryogenic temperatures using radiolytic one-electron reduction of the oxy-P450 CYP101 complex. Since a rate-limiting step in the catalytic cycle of the enzyme is the reduction of the ferrous oxygenated heme protein, subsequent reaction intermediates do not normally accumulate. Using (60)Co gamma-irradiation, the primary reduced oxy-P450 species at 77 K has been identified as a superoxo- or hydroperoxo-Fe(3+)-heme complex (Davydov, R., Macdonald, I. D. G., Makris, T. M., Sligar, S. G., and Hoffman, B. M. (1999) J. Am. Chem. Soc. 121, 10654-10655). The electronic absorption spectroscopy is an essential tool to characterize cytochrome P450 intermediates and complements paramagnetic methods, which are blind to important diamagnetic or antiferromagnetically coupled states. We report a method of trapping unstable states of redox enzymes using phosphorus-32 as an internal source of electrons. We determine the UV-visible optical spectra of the reduced oxygenated state of CYP101 and show that the primary intermediate, a hydroperoxo-P450, is stable below 180 K and converts smoothly to the product complex at approximately 195 K. In the course of the thermal annealing, no spectral changes indicating the presence of oxoferryl species (the so-called compound I type spectrum) was observed.

Cytochrome P-450 Enzyme System↗

PubMed2001

Understanding thermostability in cytochrome P450 by combinatorial mutagenesis.

The cytochromes P450 are an important class of mono-oxygenases involved in xenobiotic metabolism and steroid biosynthesis in a diverse set of life forms. Discovery of CYP-119, a P450 from the archea Sulfolobus solfataricus has provided a means for understanding nature's method of stabilizing this important protein superfamily. To identify classes of stabilizing interactions used by CYP-119, we have generated a randomized library of point mutants and screened for mutants that are less thermostable than the wild type by monitoring the characteristic Soret band in the visible region of the cell lysis. The selected mutants were characterized by differential scanning calorimetry to compare the temperatures of the melting transitions of the various mutants. The identified mutations suggested that electrostatic interactions involving salt links and charge-charge interactions, as well as contributions from other interactions such as aromatic stacking, and side chain volume of hydrophobic residues contribute to enhanced thermostability in this cytochrome P450.

Cytochrome P-450 Enzyme System↗

PubMed2000

Macromolecular hydration changes associated with BamHI binding and catalysis.

In this report, the effects of osmotic pressure on BamHI cognate binding and catalysis were investigated and compared with a previous study on EcoRI (Robinson, C. R. and Sligar, S. G. (1998) Proc. Natl. Acad. Sci. U.S.A. 95, 2186-2191). Our observation of the dependence of binding and catalytic parameters on osmotic pressure has allowed for the comparison of hydration changes associated with site-specific DNA recognition for both endonucleases. Over a large range of osmotic pressures (pi), the dependence of BamHI on osmotic stress during cognate binding and catalysis was very different from that of the related endonuclease EcoRI. The binding of EcoRI to cognate DNA was dominated by a dehydration of the endonuclease-DNA complex, whereas binding by BamHI to its cognate sequence was accompanied by a solvent release corresponding to some 125 fewer waters. Catalytic analysis at elevated osmotic pressures indicated that both endonucleases had undergone a net hydration of the complex with BamHI displaying a much greater dependence on osmotic stress than EcoRI. Although the enzymes shared core structural motifs, comparisons of high resolution x-ray structures revealed many different secondary structural features of the complexed endonucleases. The large difference in hydration changes by both BamHI and EcoRI could be attributed to these dissimilar secondary structural features, as well as the functional differences of the two endonucleases during site-specific DNA recognition.

Binding Sites↗

PubMed2000

Analyte gradient-surface plasmon resonance: a one-step method for determining kinetic rates and macromolecular binding affinities.

A novel one-step method for determining kinetic rates and equilibrium binding affinities, termed analyte gradient-surface plasmon resonance (AG-SPR) is described. A gradient maker or HPLC pump system is used to produce a gradient so that, under continuous-flow conditions, the concentration of analyte passing over the sensor surface increases linearly with time. The rate at which analyte binds to the immobilized receptors is measured by monitoring the change in the surface plasmon resonance minimum as the analyte concentration increases. Kinetic rates are determined by fitting the data to a modified version of the previously described two-compartment model (Schuck, P.; Minton, A. P. Anal. Biochem. 1996, 240, 262-272). Numerical simulations indicate that AG-SPR results in accurate estimates of both kinetic rates and equilibrium affinities regardless of the intrinsic kinetics of the interaction and can be used for systems under mass transport limitations. Simulations also indicate that AG-SPR can be used to characterize interactions that do not obey pseudo-first-order kinetics due to the presence of a heterogeneous receptor population. Experimentally, the interaction of cytochrome c with cytochrome b5 immobilized on a negatively charged monolayer has been characterized by AG-SPR, and both the specific and the nonspecific interactions were quantitatively analyzed. This new technique is advantageous over traditional SPR methods because it eliminates the need for surface regeneration and is significantly faster than traditional titration experiments.

Cytochrome c Group↗

PubMed2000

The catalytic pathway of cytochrome p450cam at atomic resolution.

Members of the cytochrome P450 superfamily catalyze the addition of molecular oxygen to nonactivated hydrocarbons at physiological temperature-a reaction that requires high temperature to proceed in the absence of a catalyst. Structures were obtained for three intermediates in the hydroxylation reaction of camphor by P450cam with trapping techniques and cryocrystallography. The structure of the ferrous dioxygen adduct of P450cam was determined with 0.91 angstrom wavelength x-rays; irradiation with 1.5 angstrom x-rays results in breakdown of the dioxygen molecule to an intermediate that would be consistent with an oxyferryl species. The structures show conformational changes in several important residues and reveal a network of bound water molecules that may provide the protons needed for the reaction.

Camphor↗

PubMed2000

Crystallization and preliminary X-ray diffraction analysis of a cytochrome P450 (CYP119) from Sulfolobus solfataricus.

CYP119 is a cytochrome P450 with a molecular weight of 43 kDa which has been isolated from the thermophilic archaeon Sulfolobus solfataricus. This enzyme is extremely stable to high temperature and high pressure. The first crystallization and preliminary crystallographic study of CYP119 is reported here. Crystals of CYP119 were obtained by the sitting-drop vapour-diffusion method using a precipitant solution containing 20%(w/v) PEG 4000 and 0.2 M sodium thiocyanate at pH 6.4. Using synchrotron radiation, the CYP119 crystal diffracted to 1.84 A resolution. It belongs to the tetragonal space group P4(3)2(1)2, with unit-cell parameters a = b = 86.17 (0.07), c = 221.11 (0.04) A, in which the numbers in parentheses describe the standard deviations. Assuming two molecules of the CYP119 per asymmetric unit, the calculated molar volume (V(m)) is 2.38 A(3) Da(-1). Bijvoet and dispersive anomalous difference Patterson maps show a clear peak corresponding to the haem irons. The complete crystallographically defined structure is currently in progress using MIR (multiple isomorphous replacement) and MAD (multiwavelength anomalous diffraction) techniques.

Archaeal Proteins↗

PubMed1999

Resonance Raman studies of cytochrome P450BM3 and its complexes with exogenous ligands.

Resonance Raman spectra are reported for both the heme domain and holoenzyme of cytochrome P450BM3 in the resting state and for the ferric NO, ferrous CO, and ferrous NO adducts in the absence and presence of the substrate, palmitate. Comparison of the spectrum of the palmitate-bound form of the heme domain with that of the holoenzyme indicates that the presence of the flavin reductase domain alters the structure of the heme domain in such a way that water accessibility to the distal pocket is greater for the holoenzyme, a result that is consistent with analogous studies of cytochrome P450cam. The data for the exogenous ligand adducts are compared to those previously reported for corresponding derivatives of cytochrome P450cam and document significant and important differences for the two proteins. Specifically, while the binding of substrate induces relatively dramatic changes in the nu(Fe-XY) modes of the ferrous CO, ferric NO, and ferrous NO derivatives of cytochrome P450cam, no significant changes are observed for the corresponding derivatives of cytochrome P450BM3 upon binding of palmitate. In fact, the spectral data for substrate-free cytochrome P450BM3 provide evidence for distortion of the Fe-XY fragment, even in the absence of substrate. This apparent distortion, which is nonexistent in the case of substrate-free cytochrome P450cam, is most reasonably attributed to interaction of the Fe-XY fragment with the F87 phenylalanine side chain. This residue is known to lie very close to the heme iron in the substrate-free derivative of cytochrome P450BM3 and has been suggested to prevent hydroxylation of the terminal, omega, position of long-chain fatty acids.

Bacillus megaterium↗

PubMed1999

Connection between the taxonomic substates and protonation of histidines 64 and 97 in carbonmonoxy myoglobin.

Infrared spectra of heme-bound CO in sperm whale carbonmonoxy myoglobin and two mutants (H64L and H97F) were studied in the pH range from 4.2 to 9.5. Comparison of the native protein with the mutants shows that the observed pH effects can be traced to protonations of two histidine residues, H64 and H97, near the active site. Their imidazole sidechains experience simple, uncoupled Henderson-Hasselbalch type protonations, giving rise to four different protonation states. Because two of the protonation states are linked by a pH-independent equilibrium, the overall pH dependence of the spectra is described by a linear combination of three independent components. Global analysis, based on singular value decomposition and matrix least-squares algorithms enabled us to extract the pK values of the two histidines and the three basis spectra of the protonating species. The basis spectra were decomposed into the taxonomic substates A(0), A(1), and A(3), previously introduced in a heuristic way to analyze CO stretch spectra in heme proteins at fixed pH (see for instance, Biophys. J. 71:1563-1573). Moreover, an additional, weakly populated substate, called A(x), was identified. Protonation of H97 gives rise to a blue shift of the individual infrared lines by about 2 cm(-1), so that the A substates actually appear in pairs, such as A(0) and A(0)(+). The blue shift can be explained by reduced backbonding from the heme iron to the CO. Protonation of the distal histidine, H64, leads to a change of the infrared absorption from the A(1) or A(3) substate lines to A(0). This behavior can be explained by a conformational change upon protonation that moves the imidazole sidechain of H64 away from the CO into the high-dielectric solvent environment, which avoids the energetically unfavorable situation of an uncompensated electric charge in the apolar, low-dielectric protein interior. Our results suggest that protonation reactions serve as an important mechanism to create taxonomic substates in proteins.

Animals↗

PubMed1999

Nature's universal oxygenases: the cytochromes P450.

Cytochromes P450 are utilized in an enormous diversity of biological reactions, including degradation of xenobiotics, generation of hormones and biosynthesis of a variety of important biological compounds. The cytochrome P450 family is a major participant in nearly all metabolism of pharmaceutical reagents. The presence of different P450 enzymes in various quantities in individuals makes the prediction of drug responses in patients highly complex. A large literature describing mechanistic studies has characterized several intermediates in the oxygenation pathway. It has recently been shown that two or more possible oxygenated forms of the P450 haem can participate in various oxygenations, with some intermediates being highly electrophilic and others being nucleophilic.

Animals↗