The structural principles of multidomain organization of the giant polypeptide chain of the muscle titin protein: SAXS/WAXS studies during the stretching of oriented titin fibres.

Elasticity of titin is a key parameter that determines the mechanical properties of muscle. These include reversibility, i.e., the muscle's capacity to change its length many-fold and return to its original state, and the transduction of passive tension generated by the stretched muscle. The morphology and elastic properties of oriented fibres of titin molecules were studied using SAXS and WAXS (small- and wide-angle X-ray scattering, respectively) and mechanical techniques. We succeeded in obtaining oriented filaments of purified titin suitable for diffraction measurements. Our X-ray data suggest a model of titin as a nanoscale, morphological, and aperiodical array of rigid Ig- and Fn3-type domains covalently connected by conformationally variable short loops. The line group symmetry of the model can be defined as SM with axial translation tau(infinity). Both tension transduction and high elasticity of titin can be explained in terms of crystalline polymer physics. Titin stretching experiments show that each individual titin macromolecule can adopt a novel two-phase state within the fibre. Conversion between high elasticity and strength can be explained as a phase transition under external tension. In the terms of the concept of orientational melting the origin of the functional heterogeneity along the titin strand becomes interpretable.

[Effect of various humidity on local dynamic structure of lysozyme in a spin-labeled tetragonal crystal]. / Lokal'naia dinamicheskaia struktura lizotsima v spin-mechenom tetragonal'nom kristalle pri izmenenii vlazhnosti.

The dynamics of the side groups of amino acid residues and local conformational changes in the lysozyme molecule upon dehydration and rehydration of lysozyme crystals were studied by the methods of spin label, X-ray diffraction, and molecular dynamics. The His15 residue of lysozyme from chicken egg white was modified by spin label, and spin-labeled tetragonal crystals of the protein were grown. The spatial structure of the covalently bound spin label and its immediate surroundings in the lysozyme tetragonal crystal was determined. The conformation of a fragment of the lysozyme molecule with the spin label on His15, optimized by the method of molecular dynamics, closely agreed with X-ray data. It was found by the X-ray diffraction analysis that a decrease in relative humidity to 40% is accompanied by both a decrease in the unit cell volume by 27% and a change in the diffraction field of roentgenograms from 0.23 to 0.60 HM. The dehydration of spin-labeled lysozyme crystals leads to an anomalous widening of EPR peaks without changes in their position. The dehydration in the humidity range studied has a two-stage character. The decrease in humidity to 75% is accompanied by a sharp change in the parameters measured, and on further decrease in humidity to 40% they change insignificantly. The first stage is caused by the removal of the greater part of molecules of bulk water, and the second stage is due to the removal of the remaining bulk water and possible changes in the dynamics of weakly bound water molecules and their position. The simulation of experimental EPR spectra showed that the anomalous broadening of the spectrum upon dehydration is related to an increase in the dispersion of spin label orientations induced by changes in the network of hydrogen bonds generated by water molecules in the vicinity of the spin label and a possible turn (by no more than 5 degrees) of the entire protein molecule. After rehydration, the physical state of the lysozyme crystal did not return to the starting point.

De novo design of fibrils made of short alpha-helical coiled coil peptides.

BACKGROUND: The alpha-helical coiled coil structures formed by 25-50 residues long peptides are recognized as one of Nature's favorite ways of creating an oligomerization motif. Known de novo designed and natural coiled coils use the lateral dimension for oligomerization but not the axial one. Previous attempts to design alpha-helical peptides with a potential for axial growth led to fibrous aggregates which have an unexpectedly big and irregular thickness. These facts encouraged us to design a coiled coil peptide which self-assembles into soluble oligomers with a fixed lateral dimension and whose alpha-helices associate in a staggered manner and trigger axial growth of the coiled coil. Designing the coiled coil with a large number of subunits, we also pursue the practical goal of obtaining a valuable scaffold for the construction of multivalent fusion proteins. RESULTS: The designed 34-residue peptide self-assembles into long fibrils at slightly acid pH and into spherical aggregates at neutral pH. The fibrillogenesis is completely reversible upon pH change. The fibrils were characterized using circular dichroism spectroscopy, sedimentation diffusion, electron microscopy, differential scanning calorimetry and X-ray fiber diffraction. The peptide was deliberately engineered to adopt the structure of a five-stranded coiled coil rope with adjacent alpha-helices, staggered along the fibril axis. As shown experimentally, the most likely structure matches the predicted five-stranded arrangement. CONCLUSIONS: The fact that the peptide assembles in an expected fibril arrangement demonstrates the credibility of our conception of design. The discovery of a short peptide with fibril-forming ability and stimulus-sensitive behavior opens new opportunities for a number of applications.

Ionic conductivity, transference numbers, composition and mobility of ions in cross-linked lysozyme crystals.

Micromethods for measurements of electric conductivity, transference numbers and concentrations of inorganic ions within immobilized protein crystals have been developed and applied to study tetragonal lysozyme crystals cross-linked with glutaraldehyde. Donnan equilibria and mobilities of ions in this crystal were calculated using the data of these methods and the data of crystal pH titration. Taken together these results characterize the lysozyme crystal as an ion exchanger whose electrical properties and ion composition differ greatly from those of the external solution. Although anions transfer most of the current in the crystals, anion mobility is considerably lower than that of cations. Mobility of all ions in the crystal is considerably lower than in solution (3.5-50 times for cations and 120-330 times for anions) and depends on steric restrictions and charges of both ions and lysozyme molecules. Similar features in behavior of crystalline and biological channels are discussed.

[X-ray fluorescent study of the ionic composition of lysozyme crystals]. / Rentgenofluorestsentnoe issledovanie ionnogo sostava kristallov lizotsima.

The method of the studying of the ionic content in the protein crystals by X-ray fluorescence spectroscopy is proposed. The ionic content of the tetragonal glutaraldehyde--cross-linked lysozyme crystals in the 2- 11 pH range and upon the low and high ionic strengths is studied. The acid-base titration of the lysozyme crystals is carried out for determination of the pH-dependence of the net charge of the lysozyme molecule within protein crystal. It has been shown that ionic content in the protein crystal channels is determined mainly by the Donnan potential. The specific binding of the bromide-ions with lysozyme molecules is revealed and possible binding sites are discussed.

[Cross-linking of crystalline sperm whale myoglobin using glutaraldehyde]. / Issledovanie fiksatsii glutarovym al'degidom molekul mioglobina kashalota v kristalle.

The salt composition of the solution has been found, which does not interact with glutaraldehyde and myoglobin at an ionic strength corresponding to that of the mother liquid, i.e., 4.5 M (NH4)2SO4. Cross-linking conditions have been elaborated when crystalline myoglobin retains its native structure: myoglobin in 3 M Cs2SO4 pH 5.4 is cross-linked by diffusion in 5% glutaraldehyde vapours in the same salt solution (5 days, room temperature).