Evaluation of the role of three candidate human kinases in the conversion of the hepatitis C virus inhibitor 2'-C-methyl-cytidine to its 5'-monophosphate metabolite.

Nucleoside analogs are effective inhibitors of the hepatitis C virus (HCV) in the clinical setting. One such molecule, 2'-C-methyl-cytidine (2'-MeC), entered clinical development as NM283, a valine ester prodrug form of 2'-MeC possessing improved oral bioavailability. To be active against HCV, 2'-MeC must be converted to 2'-MeC triphosphate which inhibits NS5B, the HCV RNA-dependent RNA polymerase.

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.

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.

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.

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.

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+.

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.

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.

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).

[Structure and regulation of the assembly of bacteriophage T4 fibrillar proteins. I. Isolation and spectral properties of long fibers].

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.

[Topology of the structural proteins of long tail fibers of phages T4D, DDVIh+ and DDVIh].

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.