Studies on calcium ion-induced conformation changes in the actin-tropomyosin-troponin system by fluorimetry. IV. Conformational changes in the region containing fluorescence-labeled sulfhydryl group(s) of troponin.

Conformational changes associated with the functional states of the molecule of troponin were studied using SH-direct fluorogenic reagents, N-(p-(2-benzimidazolyl)phenyl) maleimide (BIPM) and N-(1-anilinonaphthyl-4) maleimide (ANM). 1. The fluorescence parameters of ANM-troponin, intensity, and polarization, did not change on combining it with tropomyosin alone, but markedly changed when F-actin was further added to the system. 2. The conformation around the dye-labeled sulfhydryl group(s) was shown to be susceptible to Ca2+ in terms of fluorescence intensity of the label, thermal transition of the conformation, and the microenvironment near the label. 3. On addition of Ca2+, the fluorescence characteristics of the two systems, ANM-troponin . tropomyosin and ANM-troponin . tropomyosin . F-actin complexes, were altered in opposite directions. When BIPM was used in place of ANM, similar changes were observed: a simple decrease in the intensity when pCa was decreased from 7.4 to 5.5 in the system without F-actin and a sigmoidal increase in the range from pCa 7 to 6 in the system with F-actin. Heavy meromyosin, when added to the latter complex (the reconstituted thin filaments), made the profile of its Ca2+ concentration dependence of fluorescence similar to that of the former complex. When tropomyosin was labeled in place of troponin, similar results were obtained. The data obtained imply that the Ca2+-induced conformational changes of troponin are markedly modified when detached from actin, and that heavy meromyosin weakens the interaction of the troponin . tropomyosin complex with F-actin.     

Studies on calcium ion-induced conformation changes in the actin-tropomyosin-troponin system by fluorimetry. III. Changes in the conformation of tropomyosin associated with functional states.

The local conformational changes in the tropomyosin molecule under various conditions were studied by means of fluorimetry using SH-directed fluorescent dyes, N-(1-anilinonaphthyl-4)maleimide (ANM) and N-(3-pyrene)maleimide (PRM). 1. The fluorescence intensity, polarization and the emmission maximum of ANM-tropomyosin were found to be susceptible to ionic strength, but in different ways. The changes in these parameters suggest that the fluorescence-labeled sulfhydryl group or groups become more buried in a hydrophobic internal region by salt-induced depolymerization of aggregate and by adding F-actin to tropomyosin. 2. Titration of the labeled tropomyosin with F-actin revealed a cooperative nature in ANM labeling and a simple saturation kinetics in PRM labeling. The dissociation constant of F-actin to PRM-tropomyosin was calculated to be 5.8-10(-6) M. 3. Temperature dependence of the fluorescence polarization showed a thermal transition in the conformation of ANM- or PRM-tropomyosin at around 30 degrees C. Flexibility or segmental motion of the region containing the fluorophore was suppressed significantly on adding troponin and markedly on adding F-actin. 4. Measurements of the quantum yield and polarization of the ANM-tropomyosin-F-actin complex suggested that troponin strengthened the binding between the two proteins and that Ca2+ reversed this effect.     

Effect of phalloidin on actin proteolysis as measured by viscometry and fluorimetry.

Even at low concentration, phalloidin shows a marked protection of F-actin against the action of trypsin or pronase. G-actin is not protected at any concentration of phallodin. The kinetics of the proteolysis show that a change in the environment of tryptophan residues is preceded by disruption of the filamentous structure of F-actin.     

Studies on microplasmodia of Physarum polycephalum. VII. Adhesion-dependent changes in the organization of the fibrillar actin system

Axenically-grown microplasmodia of the acellular slime mold Physarum polycephalum were used to study adhesion-dependent changes in the spatial organization of the cytoplasmic microfilament system. Results obtained by light- and electron microscopical techniques demonstrate the presence of a membrane-bound filament cortex in all microplasmodia, and the expression of additional cytoplasmic fibrils in specimens with tight contact to a substratum. The fibrils partly terminate in focal adhesion-sites and rather seem to serve a cytoskeletal than a contractile function.     

The ion-induced folding of the hammerhead ribozyme: core sequence changes that perturb folding into the active conformation.

The hammerhead ribozyme undergoes an ion-dependent folding process into the active conformation. We find that the folding can be blocked at specific stages by changes of sequence or functionality within the core. In the the absence of added metal ions, the global structure of the hammerhead is extended, with a large angle subtended between stems I and II. No core sequence changes appear to alter this geometry, consistent with an unstructured core under these conditions. Upon addition of low concentrations of magnesium ions, the hammerhead folds by an association of stems II and III, to include a large angle between them. This stage is inhibited or altered by mutations within the oligopurine sequence lying between stems II and III, and folding is completely prevented by an A14G mutation. Further increase in magnesium ion concentration brings about a second stage of folding in the natural sequence hammerhead, involving a reorientation of stem I, which rotates around into the same direction of stem II. Because this transition occurs over the same range of magnesium ion concentration over which the hammerhead ribozyme becomes active, it is likely that the final conformation is most closely related to the active form of the structure. Magnesium ion-dependent folding into this conformation is prevented by changes at G5, notably removal of the 2'-hydroxyl group and replacement of the base by cytidine. The ability to dissect the folding process by means of sequence changes suggests that two separate ion-dependent stages are involved in the folding of the hammerhead ribozyme into the active conformation.     

Studies on cell-clearing activity in alpha-lactalbumin. Effects of calcium removal on activity and conformation.

Effects of calcium removal on the cell-clearing activity of alpha-lactalbumin (alpha-LA) and concomitant changes in conformational structure have been investigated as part of a continuing study of the activity found earlier [McKenzie, H.A. & White, F.H., Jr. (1987) Biochem. Int. 14, 347]. This activity is similar to that of lysozyme, whereby lysis of the bacterial cell wall is catalyzed. However, the specific activity of alpha-LA is on the order of 10(-6) that of lysozyme. Under conditions where activities of apo and native alpha-LA were approximately linear functions of the protein concentration, the maximal ratio of apo to native activity was 5.7:1, determined by comparison of second order velocity constants. The CD spectrum of apo alpha-LA is intermediate between that of the A state and the native protein. By NMR, the conformation of apo alpha-LA is similar to, but distinctly different from, that of the native protein. The apo form did not revert completely to the native state when Ca(II) was resupplied, consistent with a role for this cation in folding. It is suggested that the activity increase may result from a diminished constriction of the "cleft" region in alpha-LA.     

Divalent cation-, nucleotide-, and polymerization-dependent changes in the conformation of subdomain 2 of actin.

Conformational changes in subdomain 2 of actin were investigated using fluorescence probes dansyl cadaverine (DC) or dansyl ethylenediamine (DED) covalently attached to Gln41. Examination of changes in the fluorescence emission spectra as a function of time during Ca2+/Mg2+ and ATP/ADP exchange at the high-affinity site for divalent cation-nucleotide complex in G-actin confirmed a profound influence of the type of nucleotide but failed to detect a significant cation-dependent difference in the environment of Gln41. No significant difference between Ca- and Mg-actin was also seen in the magnitude of the fluorescence changes resulting from the polymerization of these two actin forms. Evidence is presented that earlier reported cation-dependent differences in the conformation of the loop 38-52 may be related to time-dependent changes in the conformation of subdomain 2 in DED- or DC-labeled G-actin, accelerated by substitution of Mg2+ for Ca2+ in CaATP-G-actin and, in particular, by conversion of MgATP- into MgADP-G-actin. These spontaneous changes are associated with a denaturation-driven release of the bound nucleotide that is promoted by two effects of DED or DC labeling: lowered affinity of actin for nucleotide and acceleration of ATP hydrolysis on MgATP-G-actin that converts it into a less stable MgADP form. Evidence is presented that the changes in the environment of Gln41 accompanying actin polymerization result in part from the release of Pi after the hydrolysis of ATP on the polymer. A similarity of this change to that accompanying replacement of the bound ATP with ADP in G-actin is discussed.     

Studies on the receptors of the MNSs group system.

Results with modified human red cell membrane sialoglycoproteins indicate that alkali-labile sialic acid and amino groups are parts of the erythrocyte receptor sites recognized by common rabbit and human anti-M and -N sera. The "N" antigen, demonstrable in MM glycoprotein preparations by rabbit anti-N, has structural properties which are similar to those of the MN receptors. Sialic acid, amino groups and carbohydrate, susceptible to periodate oxidation, are not involved in the Ss antigen sites. The specificity of the Vicia graminea lectin is dependent on free amino and carboxyl groups. Its affinity for the substances is increased by blocking of amino groups.     

The sulfhydryl groups involved in the active site of myosin B adenosinetriphosphatase. IV. Structure around the Sa thiol group.

The structure of a tryptic peptide containing one specific sulfhydryl group (Sa), which is responsible for the activation of Mg2+-ATPase of myosin B and is present in the light meromyosin region of the myosin molecule, was studied. The amino acid sequence was deduced to be Thr (or Ser)-Asn-Ala-Ala-Cys-Ala-Ala-Leu-Asp-Lys-Lys. In addition, a space-filling model around Sa was built up by comparing Sa-peptide with the amino acid sequence around Cys 190 of alpha-tropomyosin, and the high reactivity of Sa with N-ethylmaleimide is considered based on this model.