Smooth muscle alpha-tropomyosin crosslinks to caldesmon, to actin and to myosin subfragment 1 on the muscle thin filament.

To obtain proximity information between tropomyosin (Tm) and caldesmon (CaD) on the muscle thin filament, we cloned gizzard alphaTm and created two single Cys mutants S56C/C190S (56Tm) and D100C/C190S (100Tm). They were labeled with benzophenone maleimide (BPM) and UV-irradiated on thin filaments. One chain of BPM-56Tm and two chains of BPM-100Tm crosslinked to CaD. Only BPM-100Tm crosslinked to actin in the absence and presence of CaD and binding of low ratios of myosin subfragment 1 (S1) prevented the crosslinking. Tm-S1 crosslinks were produced when actin.Tm was saturated with S1. Thus, CaD on the actin.Tm filament is located <10 A away from Tm amino acids 56 and 100; in the closed state of the actin.Tm filament, Tm residue 100 is located close to the actin surface and is moved further away in the S1-induced open state; in the open state, S1 binds close to Tm.

Actin-tropomyosin activation of myosin subfragment 1 ATPase and thin filament cooperativity. The role of tropomyosin flexibility and end-to-end interactions.

Tropomyosin (Tm) bound to actin induces cooperative activation of actomyosin subfragment 1 (actin-S1) ATPase, observed as a sigmoid ATPase vs [S1] dependence. The activation is much steeper for gizzard muscle Tm (GTm) than for rabbit skeletal Tm (RSTm). To investigate if this greater cooperativity is due to increased communication between GTms along the thin filament, we studied effects of S1 binding on the state of actin-Tm using the fluorescence of pyrene-labeled Tm. Kinetic and equilibrium studies provided values for n, the apparent cooperative unit size [Geeves, M. A., and Lehrer, S. S. (1994) Biophys. J. 67, 273]. We report comparative studies of Tm-actin-S1 ATPase with values of n using GTm, RSTm, and 5aTm, a 1/7 shorter nonmuscle Tm from rat fibroblast cells [Pittenger, M. F., et al. (1994) Curr. Opin. Cell Biol., 6, 96]. 5aTm and GTm produce similar cooperative activation of actin-S1 ATPase and have similar n values that are 2-fold greater than RSTm, indicating a correlation between ATPase activation and n value. This appears to be due to the similarity of the C-terminal amino acid sequences of 5a and GTm which produce strong end-to-end interactions. The results are discussed in terms of a continuous flexible Tm strand on the actin filament.

Effects of two familial hypertrophic cardiomyopathy-causing mutations on alpha-tropomyosin structure and function.

Missense mutations in alpha-tropomyosin can cause familial hypertrophic cardiomyopathy. The effects of two of these, Asp175Asn and Glu180Gly, have been tested on the structure and function of recombinant human tropomyosin expressed in Escherichia coli. The F-actin affinity (measured by cosedimentation) of Glu180Gly was similar to that of wild-type, but Asp175Asn was more than 2-fold weaker, whether or not troponin was present. The mutations had no apparent effect on the affinity of tropomyosin for troponin. The mutations had a small effect on the overall stability (measured using circular dichroism) but caused increased local flexibility or decreased local stability, as evaluated by the higher excimer/monomer ratios of tropomyosin labeled with pyrene maleimide at Cys 190. The pyrene-labeled tropomyosins differed in their response to myosin S1 binding to the actin-tropomyosin filament. The conformations of the two mutants were different from each other and from wild-type in the myosin S1-induced on-state of the thin filament. Even though both mutant tropomyosins bound cooperatively to actin, they did not respond with the same conformational change as wild-type when myosin S1 switched the thin filament from the off- to the on-state.

Differential scanning calorimetric study of the complexes of modified myosin subfragment 1 with ADP and vanadate or beryllium fluoride.

The effects of various modifications of rabbit skeletal myosin subfragment 1 on the thermal denaturation of subfragment 1 in ternary complexes with Mg-ADP and orthovanadate (V1) or beryllium fluoride (BeFx) have been studied by differential scanning calorimetry. It has been shown that specific modifications of SH1 group of Cys-707 by different sulfhydryl reagents, trinitrophenylation of Lys-83, and reductive methylation of lysine residues promote the decomposition of the S1.ADP.Vi complex and change the character of structural transitions of the subfragment 1 molecule induced by the formation of this complex, but they have much less or no influence on subfragment 1 thermal stability in the S1.ADP.BeFx complex. Thus, the differential scanning calorimetric studies on modified subfragment 1 preparations reveal a significant difference between S1.ADP.Vi and S1.ADP.BeFx complexes. It is suggested that S1.ADP.Vi and S1.ADP.BeFx complexes represent structural analogues of different transition states of the ATPase cycle, namely the intermediate states S1**.ADP.Pi and S1*.ATP, respectively. It is also proposed that during formation of the S1.ADP.Vi complex the region containing both Cys-707 and Lys-83 plays an important role in the spread of conformational changes from the active site of subfragment 1 ATPase throughout the structure of the entire subfragment 1 molecule. In such a case, the effects of reductive methylation of lysine residues on the subfragment 1 structure in the S1.ADP.Vi complex are related to the modification of Lys-83.

Ca2+-dependent binding of calcyclin to muscle tropomyosin.

The interaction of calcyclin with tropomyosin and tropomyosin-actin was studied with fluorescence titrations and photo-reactive crosslinking experiments. Titrations of pyreneiodoacetamide-labeled tropomyosin alone or with actin showed binding of calcyclin to tropomyosin with muM dissociation constants when Ca2+ was present. UV irradiation of mixtures of calcyclin and gizzard beta beta-tropomyosin labeled with benzophenone-iodoacetamide at Cys36, with or without actin, produced crosslinks between tropomyosin chains and calcyclin monomers only in the presence of Ca2+. These data provide direct evidence for a Ca+2-dependent tropomyosin-calcyclin interaction at or near Cys36 of tropomyosin and indicate that calcyclin binding to tropomyosin-actin does not cause tropomyosin dissociation.

Kinetics of interaction of myosin subfragment 1 isoforms with F-actin.

The interaction of the myosin subfragment-1 (S-1) isoforms containing different alkali light chains A1 and A2 with F-actin has been studied using the stopped-flow technique and sedimentation in an analytical ultracentrifuge. The data obtained suggest a different mode of attachment of the S-1 isoforms to F-actin filaments.

Calorimetric characterization of the stable complex of myosin subfragment 1 with ADP and beryllium fluoride.

The thermal unfolding of the myosin subfragment 1 (S1) in its stable complex with ADP and beryllium fluoride (S1.ADP.BeF3-) was studied by differential scanning calorimetry. It has been shown that the structure of the S1 molecule in the S1.ADP.BeF3- complex is similar to that of S1 in its complex with ADP and orthovanadate (S1.ADP.Vi) but differs radically from that of nucleotide-free S1 and S1 in the S1.ADP complex. It is concluded that the S1.ADP.BeF3- complex can be considered, like the S1.ADP.Vi complex, a stable structural analogue of the myosin head.ADP.Pi transition state of the myosin-catalyzed ATP hydrolysis.

Thermal denaturation of the alkali light chain-20 kDa fragment complex obtained from myosin subfragment 1.

The thermal denaturation of the myosin subfragment 1 (S1) from rabbit skeletal muscle and of its derivatives obtained by tryptic digestion has been studied by means of differential scanning calorimetry. Two distinct thermal transitions were revealed in the isolated complex of the C-terminal 20 kDa fragment of the S1 heavy chain with the alkali light chain. These transitions were identified by means of a thermal gel analysis method. It has been shown that the thermal denaturation of the 20 kDa fragment of the S1 heavy chain correlates with the melting of the most thermostable domain in the S1 molecule. It is concluded that this domain is located in the C-terminal 20 kDa segment of the S1 heavy chain.

The effect of alkali light chains on the thermal stability of myosin subfragment 1.

Thermal denaturation of myosin subfragment 1 (S1) isoforms from rabbit skeletal muscle containing the different alkali light chains A1 and A2 [S1(A1) and S1(A2), respectively] were studied by various methods. Turbidity measurements showed that thermally induced (heating rate 1 degrees C min-1) aggregation of S1(A1) occurs at lower temperatures than that of S1(A2). However, the temperature dependences of the tryptophan fluorescence spectrum and that for ATPase inactivation were the same for S1(A1) and S1(A2). Thermal denaturation of the S1 isoforms was also studied by differential scanning microcalorimetry with the 'successive annealing' method. Three independently melting cooperative regions (domains) were revealed in the molecules of both isoforms. Heat sorption curves for the S1 isoforms were different only for the most thermolabile domain, which had a maximum at 36 degrees C for S1(A1) and at 40.5 degrees C for S1(A2). Two other peaks had maxima at 46-47 degrees C and 50-51 degrees C for both isoforms. It is proposed that alkali light chains A1 and A2 differently affect the conformation of the most thermolabile domain, which probably does not contain trytophan residues and does not take part directly in the formation of the active site of the S1 ATPase.

[Structure and regulation of the assembly of bacteriophage T4 fibrillar proteins. I. Isolation and spectral properties of long fibers]. / Struktura i reguliatsiia sborki fibrilliarnykh belkov bakteriofaga T4. I. Vydelenie i spektral'nye svoistva dlinnykh fibrill.

A preparative procedure for purification of the biological active proximal (A) and distal (BC') parts of bacteriophage T4 long-tail fibers is described. Absorption spectra of these proteins in the near ultraviolet region were measured. The absorption coefficients were determined on the basis of the nitrogen content, the absorption coefficient for the A part is epsilon 0.1% 277 nm = 0.93 +/- 0.06 and for the BC' part is epsilon 0.1%, 277,5 nm = 1.01 +/- 0.08. Calculations of the secondary structure from CD spectra show that there is a high content of beta-structure: 41% in the A part and 51% in the BC' part,--and also that alpha-helix are present in the native complex: 20% in A and 7% in BC'. A model for the spatial organisation of long fibers is proposed.

[Topology of the structural proteins of long tail fibers of phages T4D, DDVIh+ and DDVIh]. / Topologiia strukturnykh belkov dlinnykh khvostovykh fibrill fagov T4D, DDV1h+ i DDV1h.

Topology of the products of the genes 34, 35, 36 and 37 of the bacteriophage T4D long tail fibers were determined with the aid of monospecific antibodies. The antibodies against gene product 34 were the only to interact with the proximal part of long tail fibers, but the distal part bound the antibodies against 35, 36 and 37. Product of the gene 35 is located at the joint-site with the distal part and binds the distance not more than 75 A long. Gene product 36 is located between these of 35 and 37 and occupy the region about 150 A. The capability of the antibodies obtained against the above-mentioned proteins were tested ot bind with long tail fibers diagnostic phages DDVIh+ and DDVIh Shigella disentheriae. We could'nt mark any difference in binding of the antibodies against gene 34, 35 and 36 product with DDVI phages and T4D. The distal part of the fibers of DDVIh bound the antibodies against product of gene 37 as T4D. Nevertheless DDVIh+ possesses only few antigenic sites relative to product of gene 37 of T4. The changes in the distal part of long tail fibers of h-strain DDVI may lead to the broadening of the host specifity of this virus.