More about properties of Morse oscillator.

Using Birge-Sponer extrapolation we have analyzed the approximation of the potential of a real diatomic molecule by the Morse model, which implies a constant value of anharmonicity ωx. The real values of ωx*(v) for each vibrational level are estimated from transition frequencies between neighboring levels. The dependence of ωx* on the vibrational quantum number v up to dissociation is calculated from the literature data for the ground electronic state of H2, O2, Be2, Li2, ArXe, Xe2, Kr2 and the excited state of Li2. Characteristic features of deviations of the anharmonicity parameter x* - x from the Morse model are described.

Nanodiscs in the studies of membrane-bound cytochrome P450 enzymes.

Cytochromes P450 from eukaryotes and their native redox partners cytochrome P450 reductases both belong to the class of monotopic membrane proteins containing one transmembrane anchor. Incorporation into the lipid bilayer significantly affects their equilibrium and kinetic properties and plays an important role in their interactions. We describe here the detailed protocols developed in our group for the functional self-assembly of mammalian cytochromes P450 and cytochrome P450 reductases into Nanodiscs with controlled lipid composition. The resulting preparations are fully functional, homogeneous in size, composition and oligomerization state of the heme enzyme, and show an improved stability with respect to P420 formation. We provide a brief overview of applications of Nanodisc technology to the biophysical and biochemical mechanistic studies of cytochromes P450 involved in steroidogenesis, and of the most abundant xenobiotic-metabolizing human cytochrome P450 CYP3A4.

Cryoradiolysis and cryospectroscopy for studies of heme-oxygen intermediates in cytochromes p450.

Cryogenic radiolytic reduction is one of the most straightforward and convenient methods of generation and stabilization of reactive iron-oxygen intermediates for mechanistic studies in chemistry and biochemistry. The method is based on one-electron reduction of the precursor complex in frozen solution via exposure to the ionizing radiation at cryogenic temperatures. Such approach allows for accumulation of the fleeting reactive complexes which otherwise could not be generated at sufficient amount for structural and mechanistic studies. Application of this method allowed for characterizing of peroxo-ferric and hydroperoxo-ferric intermediates, which are common for the oxygen activation mechanism in cytochromes P450, heme oxygenases, and nitric oxide synthases, as well as for the peroxide metabolism by peroxidases and catalases.

Structural differences between soluble and membrane bound cytochrome P450s.

The superfamily of cytochrome P450s forms a large class of heme monooxygenases with more than 13,000 enzymes represented in organisms from all biological kingdoms. Despite impressive variability in sizes, sequences, location, and function, all cytochrome P450s from various organisms have very similar tertiary structures within the same fold. Here we show that systematic comparison of all available X-ray structures of cytochrome P450s reveals the presence of two distinct structural classes of cytochrome P450s. For all membrane bound enzymes, except the CYP51 family, the beta-domain and the A-propionate heme side chain are shifted towards the proximal side of the heme plane, which may result in an increase of the volume of the substrate binding pocket and an opening of a potential channel for the substrate access and/or product escape directly into the membrane. This structural feature is also observed in several soluble cytochrome P450s, such as CYP108, CYP151, and CYP158A2, which catalyze transformations of bulky substrates. Alternatively, both beta-domains and the A-propionate side chains in the soluble isozymes extend towards the distal site of the heme. This difference between the structures of soluble and membrane bound cytochrome P450s can be rationalized through the presence of several amino acid inserts in the latter class which are involved in direct interactions with the membrane, namely the F'- and G'-helices. Molecular dynamics using the most abundant human cytochrome P450, CYP3A4, incorporated into a model POPC bilayer reveals the facile conservation of a substrate access channel, directed into the membrane between the B-C loop and the beta domain, and the closure of the peripheral substrate access channel directed through the B-C loop. This is in contrast to the case when the same simulation is run in buffer, where no such channel closing occurs. Taken together, these results reveal a key structural difference between membrane bound and soluble cytochrome P450s with important functional implications induced by the lipid bilayer.

Chapter 11 - Reconstitution of membrane proteins in phospholipid bilayer nanodiscs.

Self-assembled phospholipid bilayer Nanodiscs have become an important and versatile tool among model membrane systems to functionally reconstitute membrane proteins. Nanodiscs consist of lipid domains encased within an engineered derivative of apolipoprotein A-1 scaffold proteins, which can be tailored to yield homogeneous preparations of disks with different diameters, and with epitope tags for exploitation in various purification strategies. A critical aspect of the self-assembly of target membranes into Nanodiscs lies in the optimization of the lipid:protein ratio. Here we describe strategies for performing this optimization and provide examples for reconstituting bacteriorhodopsin as a trimer, rhodopsin, and functionally active P-glycoprotein. Together, these demonstrate the versatility of Nanodisc technology for preparing monodisperse samples of membrane proteins of wide-ranging structure.

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.

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.

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.

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.

Thermal stability of proteins in intermolecular complexes.

A general phenomenological model is proposed for the estimation of the influence of the formation of complexes with ligands on thermal stability of proteins. In this model the reversible processes of unfolding-refolding and of association-dissociation of protein-ligand complexes and of the irreversible chemical degradation of the unfolded protein were analyzed jointly. By using certain approximations, the analytical expressions for both the thermodynamic and kinetic stabilization are obtained. Two thermodynamic and four kinetic regimes of stabilization and destabilization can exist in such system. Each thermodynamic regime appears to be compatible with three different kinetic regimes. The effect of the formation of complexes on thermodynamic and kinetic stability of the protein is determined by the degrees of binding of the ligand to the folded and unfolded protein species and by the rates of irreversible degradation of free protein and protein in complex.

Study of the structure of streptokinase conjugates with a hydrophilic vinylpyrrolidone copolymer.

The modification of streptokinase by a synthetic N-vinylpyrrolidone copolymer leads to formation of conjugates varying in structures according to the proportions of the components in the reaction medium. Based on data obtained from spectrophotometry, as well as sedimentation and diffusion analyses, it is shown that in the presence of excess protein in the reaction medium, formation of the main chain takes place via the copolymer associated with several protein globules. Under conditions of excess modifier copolymer, either single-site and/or multiple-site bonding is possible for the protein backbone, depending on the molecular weight of the copolymer. One of the models for the conjugates obtained in this manner has been corroborated by small-angle X-ray scattering data. CD spectral analyses has been performed in order to demonstrate that covalent modification does not alter the secondary structure of streptokinase in the conjugate whereas the tertiary structure undergoes local changes in conformation.

[Enzymatic and conformational stability of polymeric complexes of alpha-amylase]. / Fermentativnaia i konformatsionnaia stabil'nost' polimernykh kompleksov alpha-amilazy.

The enzymatic and conformational stability of Bacillus subtilis alpha-amylase and its polymeric complexes in acid media and subsequent renaturation in weakly alkaline media were investigated. The following parameters of alpha-amylase secondary structure were determined from circular dichroism spectra: helical units -25%, beta-structures -9%; beta-turns -13%; disordered conformations -53%. After complexation with polymethacrylic acid (PMAA) the alpha-amylase secondary structure did not change, and the tertiary structure underwent only small local changes. Complexation of alpha-amylase with linear and cross-linked PMAA led to an increase in both enzymatic and conformational stabilities in acid media. Purification of alpha-amylase using a biosorbent resulted in higher acid resistance of the free enzyme and of that in the complex with PMAA. Moreover, the degree of reversibility of the acid inactivation also increased.

[Conformation properties and enzymatic activity of pancreatic ribonuclease in soluble complexes with sodium dextran sulfate]. / Konformatsionnye svoistva i fermentaltivnaia aktivnost' pankreaticheskoi ribonukleazy v rastvorimykh kompleksakh s natrievoi sol'iu dekstransul'fata.

Effect of complex formation with dextran sulfate (DS) (substitution degree 1.3, molecular mass 500 thousand) on RNAse enzymic activity. its spatial structure and conformation stability was studied. Hydrolytic activity of the enzyme in complex in inhibited already at small additions of DS, while the transferase one is changed only at a great excess of the polyelectrolyte. It has been shown by CD spectra that no notable conformation changes proceed in the enzyme during complex formation, although the enzyme turns destabilized to the denaturing effect of heat at the expense of strengthened interactions between DS and RNAse during its denaturation. Thus the inhibition of hydrolytic activity in the complex is primarily related to limitations for the formation of the enzyme-substrate complex on polyelectrolyte charged likely with the substrate, and not to the protein conformation changes.

[Effect of polymer modification of insulin on its enzymatic hydrolysis and conformation properties]. / Vliianie polimernoi modifikatsii insulina na ego fermentativnyi gidroliz i konformatsionnye svoistva.

AI, B29 insulin polymeric derivatives in which the polymeric chains (N-polyvinylimidazole, N-polyvinylpyrrolydone and polyacrylic acid) are bonded to the insulin molecule at one point were synthesized. The hydrolysis of the modified insulin by trypsin is dependent to a great extent on the chemical nature of the modifying polymer and is virtually independent of its molecular weight up to 20 kD. The effect of the modifying polymer manifests itself mainly in a change of the Michaelis constant. Investigation of the conformational properties of the insulin derivatives by the method of optical rotatory dispersion revealed that insulin modification by polymers caused a decrease of the amino acid content in the alpha-helical sequence from 41 to 33-30%. The chemical nature of the modifying polymer and its molecular weight have a profound effect on the conformational stability of the residual spatial structure of the modified insulin in alkaline media.

[Effect of the degree of modification of trypsin amino groups with polymers on the enzymatic and conformation properties of the protein]. / Izuchenie vliianiia stepeni modifikatsii aminogrupp tripsina polimerami na fermentativnye i konformatsionnye svoistva belka.

Trypsin was modified by introducing fragments containing an azo-bond into its molecule by the reaction of free amino groups of the enzyme with an azide of 2,2'-azobisisobutryic acid. Subsequently free-radical polymerization of N-vinyl pyrrolidone was carried out with the high molecular weight initiator obtained. The degree of modification of amino groups in trypsin was n = 6 divided by 12, which distinguishes this type of modification from that earlier proposed by the authors. In that case dichlorohydrate of dimethylimidate of 2,2'-azobisisobutyric acid was used for introducing azo-bonds into the molecule of the protein, n being equal to 2-3. It is shown that under the conditions of autolytic degradation both high molecular weight initiator based on trypsin and the trypsin-PVP (poly-N-vinyl pyrrolydone) covalent conjugates exhibit higher stability than initial trypsin. The method of circular dichroism was used for comparison of conformational properties of the modified trypsin forms. An increase of the rate of thermal inactivation was found to result from conformational changes occurring on modification of the enzyme.

[Analysis of the secondary structure of urokinase by the circular dichroism method]. / Issledovanie vtorichnoi struktury urokinazy metodom krugovogo dikhroizma.

The secondary structure of urokinase with molecular weight 33 000 dalton was studied by the circular dichroism method. The secondary structure parameters were calculated based on the protein reference CD spectra resulted in the following secondary structure parameters: approximately 30% aminoacid residues constitute the alpha-helical regions, the same amount forms the beta-structure and an essential fractions contributes the beta-turns. Conformational stability of urokinase to alkaline pH (up to 11.6) and high temperature (up to 80 degrees) in 0.1 M phosphate buffer pH 6.6 was found.