Hydrophobic core malleability of a de novo designed three-helix bundle protein.

De novo protein design provides a tool for testing the principles that stabilize the structures of proteins. Recently, we described the design and structure determination of alpha(3)D, a three-helix bundle protein with a well-packed hydrophobic core. Here, we test the malleability and adaptability of this protein's structure by mutating a small, Ala residue (A60) in its core to larger, hydrophobic side-chains, Leu and Ile. Such changes introduce strain into the structures of natural proteins, and therefore generally destabilize the native state. By contrast, these mutations were slightly stabilizing ( approximately 1.5 kcal mol(-1)) to the tertiary structure of alpha(3)D. The value of DeltaC(p) for unfolding of these mutants was not greatly affected relative to wild-type, indicating that the change in solvent accessibility for unfolding was similar. However, two-dimensional heteronuclear single quantum coherence spectra indicate that the protein adjusts to the introduction of steric bulk in different ways. A60L-alpha(3)D showed serious erosion in the dispersion of both the amide backbone as well as the side-chain methyl chemical shifts. By contrast, A60I-alpha(3)D showed excellent dispersion of the backbone resonances, and selective changes in dispersion of the aliphatic side-chains proximal to the site of mutation. Together, these data suggest that alpha(3)D, although folded into a unique three-dimensional structure, is nevertheless more malleable and flexible than most natural, native proteins.

[Thermo- and photoinduced aggregation of alpha-crystallin].

Some properties of bovine alpha-crystallin, in particular its thermo- and photoaggregation, were studied by fluorescent spectroscopy of tryptophan residues and the probe 8-anilino-1-naphthalenesulfonate and light scattering. The effective diameter of a globule of native alpha-crystallin was 90 A, as estimated from the data on the polarization and lifetime of 8-anilino-1-naphthalenesulfonate using the Levshin-Perren equation, and increases at an aggregation of no less than 140 A. The increase in the intensity of tryptophan fluorescence of alpha-crystallin during its thermo- and photodenaturation with the formation of aggregates is due to local conformational changes in the surroundings of tryptophan residues and light scattering. Tryptophan residues are buried in the interior of the aggregates. The thermoaggregation of the protein occurs not only at high temperatures. By approximating the experimental time dependence of slow spontaneous aggregation to the range of large times, the time of denaturation aggregation t(e) was found. For alpha-crystallin (at a concentration of 0.8 mg/ml in phosphate buffer at pH 8.4), t(e) at 70 degrees C is 100 h. This approach can be used in finding t(e) for any protein during its thermal treatment or long-term storage.

[The globule diameter and various electron and conformational properties of the flavoprotein fragment of mitochondrial NADH dehydrogenase studied by fluorescence spectroscopy]. / Diametr globuly i nekotorye élektronno-konformatsionnye svoistva flavoproteinovogo fragmenta mitokhondrial'noi NADH-degidrogenazy po dannym fluorestsentnoi spektroskopii.

The globule dimensions and some electron and conformational properties of the flavoprotein (peripheral) fragment of the mitochondrial NADH dehydrogenase were determined by the time-resolved, phase-modulating, and polarization fluorescence spectroscopies, as well as correlated confocal microscopy. The rotational and the diffusion (translocation) diameters of the protein fragment were shown to be no less than 44 A and approximately 72 A, respectively. The diameter of protomitochondrial particles from the bovine heart, which were used for the isolation of the fraction of peripheral fragments, was no less than 2300 A. The fluorescence from tryptophan and flavin fluorophores in the fragment is strongly quenched by iron of the iron-sulfur clusters, which suggests that a strong electron-vibrational interaction of iron with Trp residues and flavin takes place. An overlapping of the electron clouds of iron-sulfur clusters, Trp residues, and flavin is likely to facilitate the electron transfer through the protein. The heat inactivation of the enzyme was accompanied by neither its substantial conformational changes, nor a considerable release of iron ions from the clusters located near the Trp residues.

[Proliferation and death of thymocytes caused by phospholipase A2 activator--melittin]. / Proliferatsiia i gibel' timotsitov pri deistvii aktivatora fosfolipazy A2--melittina.

The effects of mellitin, a component of bee venom activating phospholipase A2, on proliferation and death of the rat thymocytes were studied in a wide concentration range. Cell proliferation was estimated by the accumulation of colchicine metaphases, Necrosis was estimated by cell lysis and Trypan blue staining. Apoptosis was estimated by the type of DNA fragmentation, amount of fragmented DNA, and percentage of cells with hypodiploid DNA set. Low concentrations of mellitin (below 5 micrograms/ml) stimulated proliferation. At higher mellitin concentrations, the thymocytes die by the primary necrosis type. Mellitin did not induce apoptosis in the thymocytes within the concentration range used: on the contrary, at high concentrations, it inhibited apoptosis of the thymocytes in the control and after irradiation. Actinomycin D, inhibitor of RNA synthesis, exerted no effect on the thymocyte death in the presence of mellitin. It has been concluded that activation of phospholipase A2 may induce necrosis, rather than apoptosis, and consequently, activation of phospholipase A2 is not a necessary step in the signalling cascade that initiated apoptosis in the thymocytes.