Search PubMed⌕ Search

Biomedical subjects

J Novotný

Publications and source records attributed to J Novotný.

At least 109 records · Page 6Linked to original sources
PubMed1986

[Detection of urinary substances in stains from amniotic fluid, urine and the blood of pregnant women using thin-layer chromatography].

When examining traces of biological origin it is possible to draw conclusions from the findings of some urinary constituents in a stain produced by urine; via foetal urine, however, in the spots also amniotic fluid can be detected. By chromatographic analysis on a thin layer the authors are able to detect in spots from amniotic fluid urea without hippuric acid, in urine spots urea, creatinine and hippuric acid; in blood stains under equal conditions none of the above constituents is detected. The results, using the above method, can be considered a contribution which helps to differentiate these traces and it can be also used to detect amniotic fluid in stains.

Amniotic Fluid↗
PubMed1985

Domain association in immunoglobulin molecules. The packing of variable domains.

We have analyzed the structure of the interface between VL and VH domains in three immunoglobulin fragments: Fab KOL, Fab NEW and Fab MCPC 603. About 1800 A2 of protein surface is buried between the domains. Approximately three quarters of this interface is formed by the packing of the VL and VH beta-sheets in the conserved "framework" and one quarter from contacts between the hypervariable regions. The beta-sheets that form the interface have edge strands that are strongly twisted (coiled) by beta-bulges. As a result, the edge strands fold back over their own beta-sheet at two diagonally opposite corners. When the VL and VH domains pack together, residues from these edge strands form the central part of the interface and give what we call a three-layer packing; i.e. there is a third layer composed of side-chains inserted between the two backbone side-chain layers that are usually in contact. This three-layer packing is different from previously described beta-sheet packings. The 12 residues that form the central part of the three observed VL-VH packings are absolutely or very strongly conserved in all immunoglobulin sequences. This strongly suggests that the structure described here is a general model for the association of VL and VH domains and that the three-layer packing plays a central role in forming the antibody combining site.

Amino Acid Sequence↗
PubMed1985

Structural invariants of antigen binding: comparison of immunoglobulin VL-VH and VL-VL domain dimers.

Antigen-combining site arises by noncovalent association of the variable domain of the immunoglobulin heavy chain (VH) with that of the light chain (VL). To analyze the invariant features of the binding region (VL-VH domain interface), we compared the known immunoglobulin three-dimensional structures by a variety of methods. The interface forms a close-packed, twisted, prism-shaped "beta-barrel" characterized by cross-sectional dimensions 1.04 X 0.66 nm and a top-to-bottom twist angle of 212 degrees. The geometry of the interface is preserved via invariance of some 15 side chains, both inside the domains and on their surface. Buried polar residues form a conserved hydrogen-bonding network that has a similar topological connectivity in the two domain types; two hydrogen bonds contributed by invariant side chains extend across the interface and anchor the beta-sheets in their relative orientation. Invariant aromatic residues close-pack at the bottom of the binding-site beta-barrel with their ring planes oriented perpendicularly in the characteristic "herringbone" packing mode. Electrostatic computations that implicitly include solvent effects show the domains to be stabilized by large electrostatic forces. However, structures that were crystallized at lower pH have their electrostatic energies appropriately lowered, implying that full ionization of carboxyl side chains is essential for efficient electrostatic stabilization. The unusual mode of domain-domain association in the VL-VL dimer RHE correlates with its overall repulsive electrostatic energy (+54 kJ/mol), as opposed to negative (i.e., stabilizing) energy values (-263 to -543 kJ/mol) found in the domains of the other structures. The VL-VL dimer REI mimics closely the interface geometry of VL-VH dimers although its domain-domain contact area is lower by 18%.

Amino Acid Sequence↗
PubMed1984

An analysis of incorrectly folded protein models. Implications for structure predictions.

Proteins with homologous amino acid sequences have similar folds and it has been assumed that an unknown three-dimensional structure can be obtained from a known homologous structure by substituting new side-chains into the polypeptide chain backbone, followed by relatively small adjustment of the model. To examine this approach of structure prediction and, more generally, to isolate the characteristics of native proteins, we constructed two incorrectly folded protein models. Sea-worm hemerythrin and the variable domain of mouse immunoglobulin K-chain, two proteins with no sequence homology, were chosen for study; the former is composed of a bundle of four alpha-helices and the latter consists of two 4-stranded beta-sheets. Using an automatic computer procedure, hemerythrin side-chains were substituted into the immunoglobulin domain and vice versa. The structures were energy-minimized with the program CHARMM and the resulting structures compared with the correctly folded forms. It was found that the incorrect side-chains can be incorporated readily into both types of structures (alpha-helices, beta-sheets) with only small structural adjustments. After constrained energy-minimization, which led to an average atomic co-ordinate shift of no more than 0.7 to 0.9 A, the incorrectly folded models arrived at potential energy values comparable to those of the correct structures. Detailed analysis of the energy results shows that the incorrect structures have less stabilizing electrostatic, van der Waals' and hydrogen-bonding interactions. The difference is particularly pronounced when the electrostatic and van der Waals' energy terms are calculated by modified equations that include an approximate representation of solvent effects. The incorrectly folded structures also have a significantly larger solvent-accessible surface and a greater fraction of non-polar side-chain atoms exposed to solvent. Examination of their interior shows that the packing of side-chains at the secondary structure interfaces, although corresponding to sterically allowed conformations, deviates from the characteristics found in normal proteins. The analysis of incorrectly folded structures has made it clear that the absence of bad non-bonded contacts, though necessary, is not sufficient to demonstrate the validity of model-built structures and that modeling of homologous structures has to be accompanied by a thorough quantitative evaluation of the results. Further, certain features that characterize native proteins are made evident by their absence in misfolded models.

Amino Acid Sequence↗
PubMed1984

Twisted hyperboloid (Strophoid) as a model of beta-barrels in proteins.

Using a least-squares fitting procedure, polypeptide backbones of one parallel and seven antiparallel beta-barrels were approximated with various curved surfaces. Although the hyperboloid gave better approximations to all the beta-barrel backbones than the ellipsoid, elliptical cylinder or catenoid, the best approximations were obtained with a novel surface, a twisted hyperboloid (strophoid). The root-mean-square errors between individual beta-barrels and the fitted strophoid surfaces ranged from 0.75 A to 1.64 A. The parameters which determine the strophoid surface allow groups of beta-barrel shapes to be defined according to their barrel twists (i.e. angles subtended by directions of the long axis of cross-section at the top and the bottom of the barrel), course of elliptical cross-sections (either monotonically increasing along the barrel axis, as in cones, or having a middle "waist", as in hyperboloids), and types of backbone curvatures (either convex or concave). The curvatures at individual points of strophoid surface are local, variable quantities related to the local helicity (coil) of the polypeptide backbone, in contrast to values of beta-sheet twist (i.e. dihedral angles subtended by adjacent beta-strands) known to be virtually identical in all the beta-sheets. The variability found in parameters such as barrel shapes and curvatures suggests that simple models (isotropically stressed surfaces, principle of minimal surface tension) proposed in the past to account for beta-barrel shapes are not sufficient. Rather, the complex nature of best-fit theoretical surfaces points to an important role played by a local variability of the forces involved.

Azurin↗
PubMed1984

A program for prediction of protein secondary structure from nucleotide sequence data: application to histocompatibility antigens.

A computer program is described which, given a nucleotide or an amino acid sequence, outputs protein secondary structure prediction curves as well as hydrophobicity and charged-residue profiles. The program allows for cumulative averaging of properties (secondary structure propensities, hydrophobicity and charge profiles) from several homologous primary structures, a novel concept shown to improve the predictive accuracy. The use of the program is demonstrated on a set of nucleotide and amino acid sequences from human and murine histocompatibility antigens of class I and II. The last extracellular domains of both class I and II antigens (alpha 3 of class I, alpha 2 and beta 2 of class II) and the beta 2-microglobulin domain are predicted to consist of seven anti-parallel beta-strands, in accord with previous claims of homology between these domains and the constant domains of immunoglobulin chains. The remaining extracellular domains are all proposed to form an anti-parallel, four-stranded beta-sheet with one of its faces being covered by alpha-helices and/or structureless segments ("open face sandwiches").

Animals↗
PubMed1984

The gamma 1 heavy-chain disease FOR protein is present in two molecular forms.

Heavy chain disease proteins (FOR) were isolated from human plasma. These proteins were also detected immunochemically in the urine of the patient. The proteins were disulphide-linked Fc-like dimers with molar mass 64.2 kg/mol and sedimentation rate S020,W = 0.356 ps (3.56 S). Similar amounts of aspartic acid and pyroglutamic acid were found at the N-terminus. After cyanogen bromide cleavage of the FOR proteins, three peptides were isolated and their amino acid composition and partial amino acid sequence was determined. We suggest that two Fc-like proteins of similar sizes are present in the plasma: (1) the first with N-terminal aspartic acid corresponding to position 221 of gamma 1 EU chain and (2) the second with N-terminal pyroglutamic acid. The first protein and small amounts of related low-molar mass fragments found also in the plasma could be degradation products of the second protein. Evidence is given on structural differences between the FOR proteins and the corresponding portion of the gamma 1 EU chain.

Amino Acid Sequence↗
PubMed1983

Molecular anatomy of the antibody binding site.

The binding region of immunoglobulins, which includes the portion of the molecule having the most variability in its amino acid sequence, is shown to have a surprisingly constant structure that can be characterized in terms of a simple, well-defined model. The binding region is composed of the antigen combining site plus its immediate vicinity and arises by noncovalent association of the light and heavy chain variable domains (VL and VH, respectively). The antigen combining site itself consists of six polypeptide chain segments ("hypervariable loops") which comprise some 80 amino acid residues and are attached to a framework of VL and VH beta-sheet bilayers. Having analyzed refined x-ray crystallographic coordinates for three antigen-binding fragments (Fab KOL (Marquart, M., Deisenhofer, J., and Huber, R. (1980) J. Mol. Biol. 141, 369-391), MCPC 603 (Segal, D., Padlan, E. A., Cohen, G. H., Rudikoff, S., Potter, M., and Davies, D. R. (1974) Proc. Natl. Acad. Sci. U. S. A. 71, 4298-4302), and NEW (Saul, F. A., Amzel, L. M., and Poljak, R. J. (1978) J. Biol. Chem. 253, 585-597] we use the results to introduce a general model for the VL-VH interface forming the binding region. The region consists of two closely packed beta-sheets, and its geometry corresponds to a 9-stranded, cylindrical barrel of average radius 0.84 nm with an average angle of -53 degrees between its two constituent beta-sheets. The barrel forms the bottom and sides of the antigen combining site. The model demonstrates that the structural variability of the binding region is considerably less than was thought previously. Amino acid residues which are part of the domain-domain interface and appear not to be accessible to solvent or antigen contribute to antibody specificity.

Amino Acid Sequence↗