Biomedical subjects

I G Denisov

Publications and source records attributed to I G Denisov.

11 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↗

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

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↗

PubMed1992

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.

Hot Temperature↗

PubMed1990

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.

Circular Dichroism↗

PubMedDate not supplied

[Conformation properties and enzymatic activity of pancreatic ribonuclease in soluble complexes with sodium dextran sulfate].

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.

Circular Dichroism↗

PubMedDate not supplied

[Enzymatic and conformational stability of polymeric complexes of alpha-amylase].

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.

Bacillus subtilis↗

PubMedDate not supplied

[Effect of polymer modification of insulin on its enzymatic hydrolysis and conformation properties].

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.

Amino Acids↗

PubMedDate not supplied

[Effect of the degree of modification of trypsin amino groups with polymers on the enzymatic and conformation properties of the protein].

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.

Amines↗

PubMedDate not supplied

[Analysis of the secondary structure of urokinase by the circular dichroism method].

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.

Circular Dichroism↗