[Some properties of complexes formed by small heat shock proteins with denatured actin].

We applied different methods to analyze the effects of the recombinant wild-type small heat shock protein with an apparent molecular mass of 27 kD (Hsp27-wt) and its S15,78,82D mutant (Hsp27-3D), which mimics the naturally occurring phosphorylation of this protein, on the thermal denaturation and aggregation of F-actin. It has been shown that, at the weight ratio of Hsp27/actin equal to 1/4, both Hsp27-wt and Hsp27-3D do not affect the thermal unfolding of F-actin but effectively prevent the aggregation of F-actin by forming soluble complexes with denatured actin. The formation of these complexes occurs upon heating and accompanies the F-actin thermal denaturation. It is known that Hsp27-wt forms high-molecular-mass oligomers, whereas Hsp27-3D forms small dimers or tetramers. However, the complexes formed by Hsp27-wt and Hsp27-3D with denatured actin did not differ in their size, as measured by dynamic light scattering, and demonstrated the same hydrodynamic radius of 17-18 nm. On the other hand, the sedimentation coefficients of these complexes were distributed within the range 10-45 S in the case of Hsp27-3D and 18-60 S in the case of Hsp27-wt. Thus, the ability of Hsp27 to form soluble complexes with denatured actin does not significantly depend on the initial oligomeric state of Hsp27.

[Effect of mutation Arg9lGly on the thermal stability of beta-tropomyosin].

The mutation Arg91Gly (R91G) in beta-tropomyosin (beta-TM) is known to cause distal arthrogryposis, a severe congenital disorder of muscle tissues. To elucidate how this mutation affects the structural properties of beta-TM, the thermal unfolding of beta-TM carrying mutation Arg91Gly was compared with that of the wild type protein. It was shown by differential scanning calorimetry and circular dichroism that this point mutation dramatically decreases the thermal stability of a significant part of beta-TM (about a half of the molecule). This part of the beta-TM molecule carrying mutation R91G unfolds at approximately 28 degrees C, i.e., at a much lower temperature than the other part of the molecule, which unfolds at approximately 40 degrees C. Based on the comparison of the data obtained by differential scanning calorimetry with measurements of temperature dependence of pyrene excimer fluorescence, whose decrease reflects the dissociation of two beta-TM chains in the region of pyrene-labeled Cys-36, this thermal transition was assigned to the N-terminal part of beta-TM. Interestingly, the destabilizing effect of the mutation spreads along the coiled-coil assuming a high extent of cooperativity within this part of the beta-TM molecule.

[Calorimetric study of stable complexes of myosin subfragment I with adenosine diphosphate and phosphate analogs]. / Kalorimetricheskie issledovaniia stabil'nykh kompleksov subfragmenta 1 miozina s analogami adenozindifosfata i fosfata.

This short review is concerned with the application of the method of differential scanning calorimetry to study the conformational changes of isolated myosin head (myosin subfragment 1, S1) caused by the formation of the S1 complexes with Mg(2+)-ADP and P(i) analogues such as orthovanadate (V), aluminium fluoride (AIF4-) or beryllium fluoride (BeFx). These changes of the whole S1 molecule are reflected in a significant increase of S1 thermal stability and in a pronounced increase of the cooperativity of the thermal denaturation. Since the complexes S1-ADP-V, S1-ADP-AIF4- and S1-ADP-BeFx are stable analogues of the S1**-ADP-P(i) transition state of the S1-catalyzed ATP hydrolysis, it is concluded that DSC studies with these complexes offer a new and promising approach to investigate the structural changes which occur in the myosin head during Mg(2+)-ATPase reaction.

[The effect of modifying the lysine-83 residue on the thermal stability of myosin subfragment 1 and changes in it caused by nucleotide binding]. / Vliianie modifikatsii ostatka lizina-83 na termostabil'nost' subfragmenta 1 miozina i ee izmeneniia, vyzvaemye sviazyvaniem nukleotidov.

The effects of trinitrophenylation of lysyl residues of rabbit skeletal myosin subfragment 1 (S1) on thermal denaturation of S1 in the absence of nucleotides, in the presence of ADP and within S1 complexes with ADP and Pi analogues, orthovanadate (Vi) or beryllium fluoride (BeFx), have been studied by differential scanning calorimetry. It has been shown that lysyl trinitrophenylation significantly affects the thermal stability of S1, changes its domain structure, promotes the decomposition of S1.ADP.Vi and S1.ADP.BeFx complexes, and strongly prevents the structural changes in the S1 molecule induced by the formation of the S1.ADP.Vi complex without any effect on the thermal stability of S1 within S1.ADP and S1.ADP.BeFx complexes. It has been demonstrated that the effects of trinitrophenylation on the S1 structure are mainly due to specific modification of the epsilon-amino group of the Lys-83 residue.

[Myosin head structure]. / Struktura miozinovoi golovki.

The current data and concepts of the structural organization of the head of myosin, one of the major muscle proteins, are reviewed. The primary structure of the isolated myosin head (myosin subfragment-1) heavy chain and localization in it of sites and groups responsible for the binding and hydrolysis of ATP and myosin interaction with actin, are considered. Evidence is given of reciprocal spatial distribution of these sites and their localization on the myosin head surface. Some present-day concepts on the domain organization of the myosin head and its changes occurring during binding and hydrolysis of ATP, are discussed. A model describing the folding of the heavy and light chains in the myosin head is proposed.

[Interaction of isoforms of subfragment-1 of myosin, containing fluorescently labelled alkaline light chains, with muscle fiber actin]. / Vzaimodeistvie izoform subfragmenta-1 miozina, soderzhashchikh fluorestsentno mechennye shchelochnye legkie tsepi, s aktinom myshechnykh volokon.

Using polarization microfluorimetry, the interaction of myosin subfragment 1 (S1) isoforms containing alkali light chains A1 and A2 respectively (S1(A1) and S1(A2] with F-actin of single glycerinated rabbit skeletal muscle fibers was studied. The alkali light chains of S1 were substituted by reassociation for A1 or A2 chains modified by a fluorescent label (1.5-IAEDANS) at the single SH-group located in the C-terminus. It was found that in S1(A1) bound to muscle fiber F-actin the mobility of the fluorescent label is lower than in S1(A2). At the same time the S1(A1) and S1(A2) interaction with F-actin induces similar changes in polarized fluorescence of rhodamine linked to falloidine which, in turn, is specifically bound to F-actin. It is concluded that the both S1 isoforms bind to F-actin and produce similar effects on the conformational state of actin filaments in muscle fibers. Local differences between S1(A1) and S1(A2) seem to be due to the interaction of the N-terminus of A1 within S1(A1) with the C-terminal region of actin.

[Study of thermal denaturation of the rod part of myosin molecule by microcalorimetry and intrinsic fluorescence methods]. / Issledovanie teplovoi denaturatsii sterzhnevoi chasti molekuly miozina metodami mikrokalorimetrii i sobstvennoi fluorestsentsii.

Thermal denaturation of myosin rod has been studied by differential scanning microcalorimetry and intrinsic tryptophane fluorescence methods. Use of the sequence annealing in the calorimetric measurement allows to decompose the total thermogram of rod into four elementary bands with maxima at 42, 46.5, 50 and 57 degrees C. Fluorescence changes occur at temperatures which coincide with the first, second and fourth calorimetric peaks. Changes of the time resolved and steady state fluorescence of myosin rod were interpreted using the data on localization of tryptophan residues in the molecule. The tryptophan fluorescence of myosin rod is assumed to monitor the denaturational changes in high meromyosin and probably in the hinge region but not in the subfragment 2.

[Changes in the structural state of myosin filaments in glycerol-treated fibers of rabbit skeletal muscles induced by phosphorylation of myosin light chains]. / Izmenenie strukturnogo sostoianiia miozinovykh nitei v glitserinizirovannykh voloknakh skeletnykh myshts krolika, indutsiruemye fosforilirovaniem legkikh tsepei miozina.

Using the polarization microfluorimetry method, it was demonstrated that the increase in the degree of phosphorylation of myosin light chains (LC2) in extended single glycerinated fibers from rabbit psoas muscle changes the anisotropy of polarized fluorescence both tryptophan residue in the rod parts of the myosin molecule and the fluorescent label-N (iodoacetyl-aminoethyl)-5-naphthylamine-1-sulfonate (1,5-IAEDANS) bound to the SH1-group in myosin molecule heads. The changes in fluorescence anisotropy during LC2 phosphorylation were observed, when the measurements were performed only in the presence of 5 mM MgCl2. It was suggested that in the presence of MgCl2 the phosphorylation of LC2 associated with myosin heads changes their orientation and causes conformational shifts in the myosin filament core.

[Effect of phosphorylation of myosin light chains on the interaction of myosin minifilaments with F-actin]. / Vliianie fosforilirovaniia legkikh tsepei miozina na vzaimodeistvie miozinovykh minifilamentov s F-aktinom.

The effect of phosphorylation of light chains-2 (LC2) of rabbit skeletal muscle myosin on the interaction of myosin minifilaments with F-actin as well as on the actin-stimulated Mg2+-ATPase of minifilaments was studied. It was shown that in the absence of KCl the degree of F-actin-induced stimulation of myosin minifilament Mg2+-ATPase with phosphorylated LC2 exceeds 2-4-fold that with unphosphorylated LC2. Phosphorylation of LC2 considerably increases the rate of actin-stimulated Mg2+-ATPase reaction of myosin minifilaments but exerts only a very weak influence on the affinity of minifilaments for F-actin. After addition of KCl the differences in the actin-stimulated Mg2+-ATPase activity disappear in a great degree; in the presence of 50 mM KCl they do not exceed 50%. It was assumed that the observed specific influence of LC2 phosphorylation on the kinetic parameters of actin-stimulated Mg2+-ATPase reaction of myosin minifilaments is due to unique properties of the minifilaments (e.g., their ability to ordered self-assembly as a result of interaction between the heads of myosin molecules) which reflect their structural peculiarities.

[Phosphorylation of light chains of myosin from rabbit skeletal muscles affects the type of conformation changes of F-actin induced by heavy meromyosin]. / Fosforilirovanie legkikh tsepei miozina iz skeletnykh myshts krolika vliiaet na kharakter konformatsionnykh izmenenii F-aktina, indutsiruemykh tiazhelym meromiozinom.

The changes in F-actin conformation in myosin-free single ghost fiber induced by the binding of heavy meromyosin (HMM) with dephosphorylated or phosphorylated light chains-2 (LC2) have been studied by measuring intrinsic tryptophan polarized fluorescence of F-actin. It has been found that at low concentrations of Ca2+ (pCa greater than or equal to 8), the binding of HMM with dephosphorylated LC2 to F-actin in ghost fibres increases, whereas the binding of HMM with phosphorylated LC2 decreases the anisotropy of polarized tryptophan fluorescence. The effect is reversed at high concentrations of Ca2+ (pCa = 5). It has been assumed that this effect of myosin light chains phosphorylation may be due to its influence on the type of myosin head binding to F-actin.