Biomedical subjects

N Kurochkina

Publications and source records attributed to N Kurochkina.

6 recordsLinked to original sources

PubMed2000

Conversion of fibrinogen to fibrin: mechanism of exposure of tPA- and plasminogen-binding sites.

Conversion of fibrinogen into fibrin results in the exposure of cryptic interaction sites and modulation of various activities. To elucidate the mechanism of this exposure, we tested the accessibility of the Aalpha148-160 and gamma312-324 fibrin-specific epitopes that are involved in binding of plasminogen and its activator tPA, in several fragments derived from fibrinogen (fragment D and its subfragments) and fibrin (cross-linked D-D fragment and its noncovalent complex with the E(1) fragment, D-D. E(1)). Neither D nor D-D bound tPA, plasminogen, or anti-Aalpha148-160 and anti-gamma312-324 monoclonal antibodies, indicating that their fibrin-specific epitopes were inaccessible. The Aalpha148-160 epitope became exposed only upon proteolytic removal of the beta- and gamma-modules from D. At the same time, both epitopes were accessible in the D-D.E(1) complex, indicating that the DD.E interaction resulted in their exposure. This exposure was reversible since the dissociation of the D-D.E(1) complex made the sites unavailable, while reconstitution of the complex made them exposed. The results indicate that upon fibrin assembly, driven primarily by the interaction between complementary sites of the D and E regions, the D regions undergo conformational changes that cause the exposure of their plasminogen- and tPA-binding sites. These changes may be involved in the regulation of fibrin assembly and fibrinolysis.

Animals↗

PubMed1998

Heterogeneity of packing: structural approach.

Analysis of the heterogeneity of packing in proteins showed that different groups of the protein preferentially contribute to low- or high-density regions. Statistical distribution reveals the two preferable values for packing density in the form of two peaks. One peak occurs in the range of densities 0.55-0.65, the other occurs in the range 0.75-0.8. The high-density peak is originated primarily by high packing inside the hydrogen bonded backbone and to some extent by side chains. Polar/charged and apolar side chains both contribute to the low-density peak. The average packing density values of individual atomic groups significantly vary for backbone atoms as well as for side chain atoms. The carbonyl oxygen atoms of protein backbone and the end groups of side chains show lower packing density than the rest of the protein. The side-chain atomic groups of a secondary structure element when packed against the neighboring secondary structure element form stronger contacts with the side chains of this element than with its backbone. Analysis of the low-density regions around each buried peptide group was done for the set of proteins with different types of packing, including alpha-alpha, alpha-beta, and beta-beta packing. It was shown that cavities are regularly situated in the groove of secondary structure element packed against neighboring elements for all types of packing. Low density in the regions surrounding the peptide groups and the end groups of side chains can be explained by their positioning next to a cavity formed upon the association of secondary structure elements. The model proposed can be applied to the analysis of protein internal motions, mechanisms of cellular signal transduction, diffusion through protein matrix, and other events.

Algorithms↗

PubMed1997

Stabilization of a recombinant Fv fragment by base-loop interconnection and V(H)-V(L) permutation.

We have developed a novel method to stabilize a recombinant antibody Fv fragment. The V(H) and V(L) domains of this Fv fragment, called pFv (permutated Fv), are covalently interconnected to each other at the two "base-loops" that normally connect V(H) beta strand 3 to 3b and V(L) beta strand 3 to 3b. To produce the base-loop stabilized Fv fragment, we connected the N-terminal half of the V(L) domain (V(L) 1-40) of murine antibody anti-Tac to the C-terminal half of V(H) (V(H) 42-115). We also fused the C terminus of V(H) by a (Gly4Ser)3 linker to the N-terminal half of V(H) (V(H) 1-40, thereby generating a permutated V(H) domain). Finally we connected the base loop of V(H) (N-terminal half) to the C-terminal half of V(L) (V(H) 42-115). The anti-Tac pFv fragment was fused to a truncated form of Pseudomonas exotoxin to generate a pFv-immunotoxin. Fvs with the correct structure were produced by refolding of recombinant inclusion body protein using a renaturation protocol that was originally developed for Fab and scFv fragments. Due to the artificially connected and permutated primary sequence, the folding pathway for the pFv structure may possibly be different from the conventional folding of antibody domains. Analysis of antigen binding of anti-Tac pFv, and of the specific cytotoxicity of pFv-immunotoxin towards antigen expressing cancer cells demonstrated that the anti-Tac pFv retained most of its affinity and full specificity when compared to anti-Tac scFv. Also anti-Tac pFv was relatively stable, retaining 25% of its binding activity after a 24 hour incubation in human serum at 37 degrees C. This indicates that connection of base loops can be a useful alternative to linker or disulfide stabilization of Fv fragments.

Amino Acid Sequence↗

PubMed1996

Protein folding by a biased Monte Carlo procedure in the dihedral angle space.

A straightforward method for predicting the protein structure is to find conformations that have the lowest energy along a chosen folding pathway. One approach in this direction is to produce a large number of structures by varying the dihedral angles of the molecule more or less randomly and then to screen each one using a suitable energy function. This procedure is computationally demanding, but by using a more realistic model, one hopes that the folding behavior one observes in calculations may better mimic the actual folding process in nature. The method is beginning to yield interesting results, thanks to the increase in the computational power but also to the intelligent selection of the folding pathway. This article reviews general features of this method, some important highlights of the particular procedure we used, and some of the more significant results obtained to date in our laboratory. The results are highly encouraging and indicate the direction of future effort that is most likely to be fruitful.

Computer Simulation↗

PubMed1995

Hydrophobic potential by pairwise surface area sum.

An approximate but rapid method for estimating hydrophobic energy is proposed. Aside from a scale factor, it is given by the pairwise sum of the surface area buried by each neighbor atom, but excluding those atoms in the same residue or in its sequence neighbor residues. This sum is found to be linearly related to the true buried area as calculated by the algorithm of Lee and Richards [1971, J. Mol. Biol., 55, 379-400], and to the contact potential of Miyazawa and Jernigan [1985, Macromolecules, 18, 534-552]. It correlates with experimental transfer free energies to approximately the same degree as that calculated using the true buried area. Furthermore, in a simple test of helix packing with ROP protein monomer, the new hydrophobic energy clearly discriminated one structure, with the lowest r.m.s. deviation from the crystal structure, against an exhaustive set of others.

Amino Acids↗

PubMed1989

Molecular cartography of proteins: surface relief analysis of the calf eye lens protein gamma-crystallin.

Methods of calculating the protein molecular surface and different map representations are described. The maps are obtained by projection of the space-filling molecular model on the surface of the ellipsoid of inertia. A new approach to surface analysis is proposed which is based on the use of three general maps: an identification map with all residues outlined, a surface relief map and a coloured map with a specific colour for each of the surface atoms. Superposition of these maps greatly simplifies molecular surface analysis. The usefulness of such an approach has been demonstrated by the study of the relief of the calf eye lens protein gamma-crystallin II. Protrusions of the relief have been shown to be occupied generally by charged residues, but in some cases by the hydrophobic ones. It is interesting to note that in crystal medium the protruding residues are involved, in the majority of cases, in intermolecular contacts. The protruding regions have been found to be pseudosymmetrical to each other in accordance with the two-fold rotation axis of the molecule. However, the colours of these regions, i.e. the atoms of the corresponding side chains, differ greatly.

Animals↗