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E A Permyakov

Publications and source records attributed to E A Permyakov.

53 records · Page 3Linked to original sources
PubMed1985

Effects of cation binding on the thermal transitions in calmodulin.

The thermal transitions in different forms of bovine brain calmodulin (0, 1, 2, 3 and 4 bound Ca2+ ions per molecule) have been studied by means of microcalorimetry, intrinsic tyrosine fluorescence, circular dichroism and infrared spectroscopy. The heating of the apoprotein from 5 to 110 degrees C induces at least three unfolding transitions. The heating of Ca2+-loaded calmodulin causes at least two structural transitions, one of which occurs at relatively low temperatures, from approx. 30 to approx 50 degrees C. The binding of the biologically significant Ca2+, Mg2+, Na+ and K+ ions has been measured at 12, 20, 28, 37 and 50 degrees C by means of the fluorescence method. The values of the binding parameters for these cations do not depend on temperature within the range 12 to 50 degrees C. It has been proposed that the temperature independence of the metal-ion-binding properties of calmodulin is achieved due to the temperature-induced structural changes, which adjust the protein conformation in such a way that the protein-binding parameters remain constant.

Animals↗
PubMed1985

Parvalbumin conformers revealed by steady-state and time-resolved fluorescence spectroscopy.

The binding of calcium to whiting (one tryptophan residue) and pike (one tyrosine residue) parvalbumins has been studied by means of kinetic and steady-state fluorescence techniques. The decay curves of the tryptophan and tyrosine fluorescence of the parvalbumins are best fitted by a sum of two exponents for any metal state of the proteins. The data can be interpreted as a nonexponential decay of the fluorescence of a single-type chromophore or in terms of equilibria between compact and relaxed conformers of the parvalbumins in each metal state. Fluorescence quenching by I-ions and effects of H2O/D2O substitution confirm the second interpretation. The constants of the equilibria have been evaluated.

Animals↗
PubMed1985

Cation binding effects on the pH, thermal and urea denaturation transitions in alpha-lactalbumin.

The binding of monovalent (Na+, K+) and divalent (Ca2+, Mg2+) cations to bovine alpha-lactalbumin at 20 and 37 degrees C has been studied by means of intrinsic protein fluorescence. The values of apparent binding constants for these ions obtained at 37 degrees C are about one order of magnitude lower than those measured at 20 degrees C. Urea and alkali (pH greater than 10) induce unfolding transitions which involve stable partially unfolded intermediates for all metal ion-bound forms of alpha-lactalbumin. Heating induces similar partially unfolded states. Nevertheless, the partially unfolded states induced by heating, urea, alkaline or acidic treatments are somewhat different in their tryptophan residue environment properties. The results have been interpreted in terms of a simple scheme of equilibria between metal-free and metal-bound forms in their native, partially unfolded and unfolded states. The scheme provides an approach to the quantitative interpretation of any transition equilibrium shift induced by a low molecular mass species able to be bound by a protein.

Animals↗
PubMed1984

Some aspects of studies of thermal transitions in proteins by means of their intrinsic fluorescence.

The changes in intrinsic fluorescence parameters induced by thermal transitions in proteins are developed on the background of the common thermal fluorescence quenching due to an activation of collisions between the excited chromophores and neighbouring quenching groups. Two methods of separation of the thermal quenching and conformational change contributions to the temperature dependence of the fluorescence parameters are presented. One is based on the use of the linearity of the plots of the reciprocal fluorescence quantum yield, 1/q, vs. the T/eta ratio (T, temperature; eta, solvent viscosity) for native proteins containing a single fluorescing chromophore (T.L. Bushueva , E.P. Busel and E.A. Burstein , Biochim. Biophys, Acta 534 (1978) 141). The other method is based on a consideration of the phase plots for the tryptophan fluorescence of proteins (fluorescence intensity at a fixed wavelength vs. intensity at any other fixed wavelength). The methods have been used for a study of the thermal transitions in Mg2+-loaded whiting parvalbumin (tryptophan fluorescence), Mg2+-loaded pike parvalbumins pI 4.2 (tyrosine fluorescence) and pI 5.0 (phenylalanine fluorescence), and Ca2+-loaded bovine alpha-lactalbumin (tryptophan fluorescence). The thermal denaturation curves for the parvalbumins show two-stepped character. The main change of the protein conformation occurs at the higher temperature step. Comparison of the fluorescence data with the microcalorimetry results shows that the maxima of the asymmetric heat sorption peaks for pike parvalbumins correlate with the mid-points of the higher temperature steps of the fluorimetric curves.

Animals↗
PubMed1983

Sodium and potassium binding to parvalbumins measured by means of intrinsic protein fluorescence.

The binding of Na+ and K+ to whiting parvalbumin (pI 4.4) and pike parvalbumins (pI 4.2 and 5.0) results in a shift of the tryptophan fluorescence spectrum towards shorter wavelengths by 2-4 nm for the whiting protein and in a rise of the tyrosine and phenylalanine fluorescence quantum yield for the pike proteins. The effective binding constants of Na+ and K+ to parvalbumins are within the range of 10 M-1 to 100 M-1. Physiological concentrations of Na+ and K+ lower the affinity of whiting parvalbumin for Ca2+ and Mg2+ by almost an order of magnitude.

Animals↗
PubMed1983

Comparative study of physiochemical properties of two pike parvalbumins by means of their intrinsic tyrosyl and phenylalanyl fluorescence.

Physicochemical properties of two pike parvalbumins (pI 5.0 and 4.2), belonging to two different gene lineages, have been studied by their intrinsic tyrosine and phenylalanine fluorescence. The CD sites of these paravalbumins have similar affinities to Ca2+ (and Mg2+) ions, but the EF sites of the proteins have very different affinities to these ions. This results in differing stabilities of these parvalbumins to pH-, urea-, and temperature-induced denaturation. The structure of pike parvalbumin pI 5.0, which binds Ca2+, and Mg2+ ions more tightly, is more stable than that of parvalbumin pI 4.2. Both proteins have higher affinities for Na+ ions than for K+ ions.

Animals↗
PubMed1983

A spectrofluorometric study of the environment of tryptophans in bacteriorhodopsin.

The emission spectrum of intact purple membranes of Halobacterium halobium has a very short wavelength position (the main maximum at 314 nm) and can be fitted by two spectral components, one of which (component A) corresponds to the fluorescence of buried tryptophan residues located in a highly hydrophobic rigid environment (like the single tryptophan residue in azurin), the other (component I) being due to the emission of buried tryptophan residues located in a rather polar environment. Treatment of bacteriorhodopsin by NaBH4, fragmentation of the membranes and thermal formation of vesicles result in a decrease in the contribution of component A, an increase in that of component I and the appearance of spectral components corresponding to the emission of surface tryptophan residues. Temperature induces at least two distinct changes of the fluorescence parameters of the protein: one change occurs from 45 to 65 degrees C. the other from 65 to 90 degrees C. The spectral changes correlate with the peaks of heat sorption caused by thermal transitions in the purple membrane structure and conformational changes in the protein structure. Alkaline denaturation of bacteriorhodopsin registered by tryptophan fluorescence begins at pH > 11.0.

Journal Article↗
PubMed1982

Intrinsic fluorescence spectra of a tryptophan-containing parvalbumin as a function of thermal, pH and urea denaturation.

The thermal, pH and urea denaturation of the calcium-loaded protein from whiting has been studied by means of the intrinsic fluorescence of the single tryptophan residue. pH denaturation of the protein takes place at a pH greater than 11.5 and lower than 5.5. Thermal denaturation of the protein occurs at temperatures above 55 degrees C. Urea initiates the denaturation of the calcium-loaded protein at rather low concentrations (1.0 M). In all cases, whether pH, thermal or urea denaturation, intermediate states of the protein were recorded. The fluorescence spectra of these intermediates are similar to that of the protein with one equivalent of calcium bound. Whiting parvalbumin binds calcium in the presence of 7.5 M urea but under these conditions, calcium-binding constants of the protein have been shown to be 10(2)-10(3)M(-1) (in comparison with 5 X 10 (8) and 6 X 10(6)M(-1) in the absence of any denaturing agents).

Animals↗
PubMed1980

Fluorescence studies of the calcium binding to whiting (Gadus merlangus) parvalbumin.

The calcium binding by parvalbumin of whiting (Gadus merlangus) has been studied using tryptophanyl fluorescence characteristics. Titration of Ca2+-free parvalbumin with Ca2+ leads to a very pronounced blue shift, narrowing and intensification of the fluorescence spectrum. These spectral changs proceed in two stages reflecting the existence of at least three forms which can be interpreted as (a) the protein without Ca2+, (b) with one Ca2+ and (c) with two bound Ca2+ ions/molecule. The fluorescence of these forms has been identified and the fluorescence spectra measured at varied Ca2+ concentrations were resolved into three components corresponding to these spectral forms. The dependence of the relative concentration of the three fomrs on Ca2+ concentrations agree well with the two-step binding of Ca2+ to parvalbumin: Protein + Ca in equilibrium K1 protein x Ca; Protein x Ca + Ca in equilibrium K2 Ca x protein x Ca. The equilibrium binding constants K1 and K2 obtained by the computer fit are approximately 5 X 10(8) M-1 and 6 X 10(6) M-1. This scheme and the K1 and K2 value are in a good agreement with the independent experimental data resulting from EGTA titration of Ca2+-saturated parvalbumin and pH titratin of parvalbumin in the presence of EGTA and CA2+.

Animals↗
PubMed1977

The fine structure of luminescence spectra of azurin.

The spectra of azurin absorption, fluorescence, phosphorescence and fluorescence excitation have been measured in aqueous solutions at ordinary and liquid nitrogen temperatures. The fluorescence spectra of azurin even at ordinary temperatures have a well resolved fine vibrational structure. The frequency analysis reveals practically the same wave number distances between the main structure peaks in fluorescence spectra at room and low temperatures and in phosphorescence spectra. The comparison of the protein absorption and excitation spectra shows that all the energy absorbed by tyrosine residues is transferred onto indole chromophore. These data suggest an unusual tryptophan environment in this protein, which is characterized by the absence of any hydrogen bonding or other polar interaction of tryptophan with its environment. The problem of the possibility of contributions of two electronic transitions (1La in equilibrium A and 1Lb in equilibrium A) in absorption and emission spectra of azurin tryptophan arising from their mirror symmetry is discussed.

Azurin↗
PubMed1976

Reinterpretation of luminiscence properties of neurotoxins from the venom of Middle-Asian corba Naja oxiana eichw.

A new interpretation of previous work (Bukolova-Orlova, T. G., Burstein, E.A. and Yukelson, L. Ya (1974) Biochim. Biophys. Acta 342, 272-280) and some new data on the luminescence of neurotoxins I and II from Naja oxiana venom is given, based on the newer data on their complete amino acid sequences. Very effective excitation energy exchange exists between Trp-27 and Trp-33 in neurotoxin I and between Trp-27 and Trp-28 in neurotoxin II, Which results in the tryptophanyl fluorescence spectra of each of the proteins seeming to be monocomponent ones. The lowered fluorescence quantium yield value, the shortened phosphorescence lifetime (80% of the emission has tau p less than 0.5 s, 20% has tau p = 4.8 s, comparing with usual tau p = 5.5-5.9 s) and decreased phosphorescence to fluorescence ratio (0.042, as compared to the usual 0.4-0.7) for neurotoxin I suggest that the indole chromophore of Trp-27 and/or Trp-33 are in contact with heavy sulfur atoms of disulfide, most probably of Cys(28)-Cys(32). Tryptophanyls in neurotoxin II are exposed to the solvent, however their accessibility in relation to that of the free tryptophan to the negatively charged quencher I- (0.455) is much higher than that for the positively charged Cs+ (0.08), which is probably due to the proximity of cationic Lys-25, Lys-26 and His-31. The difference of accessibility to the negative and positive quenchers is even more pronounced in the case of the neurotoxin I (1.04 and 0 +/- 0.02, respectively), though in its chromophore vicinity along the primary structure there is only one cationic group, Lys-25. This fact together with the analysis of the amino acid sequence, suggest that the space folding of this polypeptide results in the close proximity of Trp-27 and/or Trp-33 with the C-terminal peptide segment 67-73, which contains four cationic groups (His-67, Lys-69, Lys-71 and Arg-72).

Amino Acid Sequence↗
PubMed1975

Investigation of some physico-chemical properties of muscular parvalbumins by means of the luminescence of their phenylalanyl residues.

The influence of pH, temperature and Ca2+-release on the phenylalanyl and tyrosyl fluorescene of muscular parvalbumins from white muscles of hake and carp has been investigated. Within the pH range from 7 to 8, Ca2+-saturated parvalbumins show a conformational change registered by fluorescence, that is associated with the release of some of the bound Ca2+. Removal of Ca2+ by means of EGTA (ethyleneglycolbis-(aminoethylether)tetra-acetic acid) considerably narrows the region of protein nativity, increases the accessibility of their chromophores to quencher ions (Cs+ and CNS-) and decreases their stability against heat denaturation. The usefulness of measurements of the phenylalanine fluorescence and of the tyrosine-phenylalanine energy transfer in the investigation of these and other proteins is discussed.

Albumins↗
PubMedDate not supplied

Interactions of parvalbumins with model phospholipid vesicles.

Interactions of Ca2+-binding proteins, parvalbumins, with model vesicles formed with both synthetic (dipalmitoylphosphatidylcholine) and natural (phosphatidylcholine and phosphatidylethanolamine) phospholipids have been revealed and studied by means of gel-chromatography, electron microscopy, intrinsic fluorescence and microcalorimetry methods. There are at least two populations of liposome-bound parvalbumin one of which has higher affinity to the liposomes (effective binding constant about 10(6) M-1) than the other one. The interaction is modulated by Ca2+ and Mg2+ ions and induces changes in properties of both parvalbumin and liposomes.

Animals↗