V. P. Timofeev

Spin-label study of segmental flexibility of anti-hapten antibodies. Precipitating pig anti-Dnp antibody is more flexible than non-precipitating.

Relative freedom of rotation of the Fab-parts (Fab-arms flexibility) is very important for functional activity of the antibody molecules [ 11. For example, cleavage of the inter-heavy chain disulfide bonds that results in conversion of incomplete rabbit antibodies into direct hemagglutinins points to an important role of Fab-parts movement [2]. In [3] we found by a fluorescence polarization method that precipitating pig anti-Dnp antibodies, randomly labeled by dansylchloride, had a more flexible structure than nonprecipitating antibodies with the same specificity. The segmental flexibility of macromolecules can be determined by the spin-label method also [4]. Recently, a variation of that method has been described that permits evaluation of rotation of a spin-label relative to a macromolecule [5]. Using this method, the rotational correlation times i of immunoglobulin molecules and their fragments were determined [6] and found to be in fair agreement with the results of fluorescence polarization experiments. In this study, we determined the 7 values of the early and late pig anti-Dnp antibodies with precipitating and non-precipitating properties. The spinlabel was found to be attached both covalently to the protein moiety outside the combining site and noncovalently to the antibody combining site. Both these forms of label yielded identical rotational correlation times, showing that the precipitating anti-Dnp anti-

Simulation of EPR spectra of the radical TEMPO in water-lipid systems in different microwave ranges

A novel interpretation of EPR spectra of the TEMPO radical in water-lipid media in terms of the model of two independent movements of nitroxyl is presented. A program of the theoretical modeling of EPR spectra has been developed, and the results of its operation have been demonstrated by an example of well known sets of experimental EPR spectra in various phospholipid media and membranes.

Novel side chain modified Δ8(14)-15-ketosterols

Abstract Synthesis of five novel Δ8(14)-15-ketosterols comprising modified side chains starting from ergosterol is described. Ergosteryl acetate was converted into (22 E )-3β-acetoxy-5α-ergosta-8(14),22-dien-15-one through three stages in 32% overall yield; further transformations of the product obtained led to (22 E )-3β-hydroxy-5α-ergosta-8(14),22-dien-15-one, (22 S ,23 S )-3β-hydroxy-22,23-oxido-5α-ergost-8(14)-en-15-one, (22 R ,23 R )-3β-hydroxy-22,23-oxido-5α-ergost-8(14)-en-15-one, (22 R ,23 R )-5α-ergost-8(14)-en-15-on-3β,22,23-triol and (22 R ,23 R )-3β-hydroxy-22,23-isopropylidenedioxy-5α-ergost-8(14)-en-15-one. New Δ8(14)-15-ketosterols were evaluated for their cytotoxicity and effects on sterol biosynthesis in human hepatoma Hep G2 cells in comparison with known 3β-hydroxy-5α-cholest-8(14)-en-15-one. Among the compounds tested, (22 R ,23 R )-3β-hydroxy-22,23-oxido-5α-ergost-8(14)-en-15-one was found to be the most potent inhibitor of sterol biosynthesis (IC 50 xa0=xa00.6xa0±xa00.2xa0μM), whereas (22 R ,23 R )-5α-ergost-8(14)-en-15-on-3β,22,23-triol exhibited the highest cytotoxicity (TC 50 xa0=xa012xa0±xa03xa0μM at a 24xa0h incubation).

Synthesis of secasterol and 24-episecasterol and their toxicity for MCF-7 cells

The convergent synthesis of biosynthetic precursors of brassinosteroids with a Δ2-bond in cycle A—secasterol and 24-episecasterol—was performed. The key stages in the construction of the side chain in these compounds were the Julia olefination of the steroid 22-aldehyde followed by the Sharpless asymmetric dihydroxylation of the intermediate Δ22-olefin. The cytotoxicity of the synthesized compounds for breast carcinoma MCF-7 cells was assessed.

Conformational changes of aspartate aminotransferases in the region of Cys-45 residue observed by means of spin label.

Abstract Two thiol groups located at the surface of the aspartate aminotransferase molecule ( l -aspartate:2-oxoglutarate aminotransferase, EC 2.6.1.1) can be alkylated by a paramagnetic label, viz. 2,2,6,6-tetramethylpiperidine-1-oxyl-4-iodoacetate. The modified enzyme restores the activity almost completely. Under the conditions used the spin label is not bound by any other functional groups of the aspartate aminotransferase molecule. Binding of ligands (substrates and quasisubstrates) at the enzyme active site induces changes of the EPR spectrum, which are reversible: removal of ligands by gel filtration through a Sephadex G-25 column results in the disappearance of these changes. One of the accessible thiol groups (Group I) was selectively alkylated by maleic acid whereas the other one (Group II) was labeled with the spin label. In this case addition of ligands did not affect the mobility of the spin label. It can be concluded, therefore, that it is only the spin label bound to the Group I that is sensitive to the interaction of a ligand with the active site. This group in the native enzyme was labeled with [14C]maleic acid. Chromatography of the chymotrypsin digest of the treated protein yielded radioactive peptide; thereafter its partial structure was identified. The amino acid sequence of the peptide corresponds to that of the enzyme region from 41 to 48 (Trp residue in the peptide under study was destroyed during acid hydrolysis). Previously it has been shown that the residue Tyr-40 is located in the region of the enzyme active site. The localization of the cysteine under study (Cys-45) makes it possible to think that this amino acid residue is rather close to the active site of the aspartate aminotransferase. However, the distance ( >17 A ) between this residue and the active site as seen from the data obtained is not sufficient to provide spacial overlapping between the spin label and the Schiffs base of coenzyme with the substrate molecule. Therefore, the effect of substrate upon the mobility of the spin label is mediated by conformational changes in a certain region of the active site.

Application of spin-labeled vitamin B6 analogues for the study of aspartate aminotransferase.

For elucidation of the chemical nature and functional role of amino acid residues in an enzyme’s active site, valuable information can be obtained by introducing a coenzyme analogue which carries either a reactive or ‘reporter’ group sensitive to its immediate surroundings. This role can be played by a spin label, e.g., an iminoxyl radical. Use of spinlabeled inhibitors and coenzyme analogues has proved an adequate approach to investigations of the topography of active sites of some enzymes [l] . In this work, the following vitamin Be analogues were used (see scheme I).

Nucleotide-induced and actin-induced structural changes in SH1-SH2-modified myosin subfragment 1

We compared the structural properties of myosin subfragment 1 (S1) modified at both reactive SH-groups, SH1 (Cys707) and SH2 (Cys697), with the properties of unmodified S1 and SH1-modified S1. It is shown using differential scanning calorimetry (DSC) that SH1 modification has no noticeable influence on the changes in S1 thermal unfolding induced by the formation of S1 ternary complexes with ADP and Pi analogs (Vi, AlF4−, and BeFx). These changes, however, normally expressed in a significant increase of S1 thermal stability, are almost fully prevented by modification of both SH1 and SH2. In contrast, SH2 modification had no effect on the changes induced by the formation of the ternary complexes S1-ADP-Vi, S1-ADP-AlF4−, and S1-ADP-BeFx in EPR spectra of S1 spin-labeled at SH1 group. Interaction of S1 with F-actin substantially increased the thermal stability of S1; a similar effect was observed by DSC with both SH1- and SH1-SH2-modified S1. Overall, our results demonstrate that modification of both reactive SH-groups on S1 has no influence on the actin-induced changes of S1 and on the local nucleotide-induced conformational changes in the SH1 group region, but strongly prevents the global nucleotide-induced structural changes in the entire S1 molecule. The results suggest that modification of SH1 and SH2 impairs the spread of nucleotide-induced conformational changes from the ATPase site throughout the structure of the entire S1 molecule, thus disturbing a coupling between the motor and regulatory domains in the myosin head.

[76] Spin-labeled vitamin B6 derivatives: Synthesis and interaction with aspartate aminotransferase

Publisher Summary Spin-labeled analogs of coenzymes, substrates, and prosthetic groups have proved to be a useful tool for elucidating structure and conformational changes in proteins. This chapter describes the synthesis of spin-labeled vitamin B 6 derivatives and their interaction with aspartate aminotransferase. The iminoxyl group of spin-labeled derivatives has been used not only as a “reporter” group but also as a specific oxidant of the cysteine residue. The chapter describes the method in which the paramagnetic analogs of pyridoxal 5-phosphate (PLP) and pyridoxamine 5-phosphate (PMP) are applied for studying AAT. The apoenzyme of AAT (apo-AAT) is stable enough and capable of binding an analog of the coenzyme. This results in a typical maximum in the circular dichroism (CD) spectrum, the appearance of which should be attributed to the asymmetrical surroundings of the chromophore. The interaction of the apo-AAT with certain paramagnetic analogs is also accompanied by a characteristic Cotton effect in the absorption band for analogs.

Pregna-5,17(20)-dien-21-oyl amides affecting sterol and triglyceride biosynthesis in Hep G2 cells

Synthesis of series [17(20)Z]- and [17(20)E]-pregna-5,17(20)-dien-21-oyl amides, containing polar substituents in amide moiety, based on rearrangement of 17α-bromo-21-iodo-3β-acetoxypregn-5-en-20-one caused by amines, is presented. The titled compounds were evaluated for their potency to regulate sterol and triglyceride biosynthesis in human hepatoma Hep G2 cells in comparison with 25-hydroxycholesterol. Three [17(20)E]-pregna-5,17(20)-dien-21-oyl amides at a concentrations of 5 μM inhibited sterol biosynthesis and stimulated triglyceride biosynthesis; their regulatory potency was dependent on the structure of amide moiety; the isomeric [17(20)Z]-pregna-5,17(20)-dien-21-oyl amides were inactive.

Structural features of Fab fragments of rheumatoid factor IgM-RF in solution

The structural features of the Fab fragments of monoclonal (Waldenström’s disease) immunoglobulin M (IgM) and rheumatoid immunoglobulin M (IgM-RF) were studied by a complex of methods, including small-angle X-ray scattering (SAXS), electron spin resonance (ESR), and mass spectrometry (MS). The Fab-RF fragment was demonstrated to be much more flexible in the region of interdomain contacts, the molecular weights and the shapes of the Fab and Fab-RF macromolecules in solution being only slightly different. According to the ESR data, the rotational correlation time for a spin label introduced into the peptide sequence for Fab is twice as large as that for Fab-RF (21±2 and 11±1 ns, respectively), whereas the molecular weights of these fragments differ by only 0.5% (mass-spectrometric data), which correlates with the results of molecular-shape modeling by small-angle X-ray scattering. The conclusion about the higher flexibility of the Fab-RF fragment contributes to an understanding of the specificity of interactions between the rheumatoid factor and the antigens of the own organism.