No abstract

By reacting trimethylammoniobromobimane bromide (TMB bromide) with rabbit muscle actin, a fluorescent reporter group was linked to cysteine at position 374. Fluorescence of TMB-actin decreased significantly on addition of thymosinp4 (T/94), a peptide of 43 amino acid residues reported to bind to monomeric actin and to prevent filament formation, Based on this effect, we determined the Kn value of the thymosin @l complex as 0.8 PM, a value that is in agreement with previous determinations. In addition to the main compound thymosin/?4, bovine tissue contains a related peptide, thymosin p (Tp9), which has 41 amino acid residues and ca. 75% sequence homology. In the present study we show for the first time that T/$?, similar to Tp4, forms a 1:l complex with monomeric actin, and hereby inhibits actin polymerization. With a K, value of 1.1 FM the affinity of TjT9 is in the same range as that of Tp4, suggesting that T@9, like T/?4, contributes to maintaining the pool of monomeric. actin in bovine non-muscle cells. Further proof of the interaction of T/I9 with actin was provided by native PAGE, where the complex showed the reported higher mobility, as well as by crosslink~g experiments. Using different crosslinking reagents, like water-soluble ~~iimide (EDC), ~-maleimido~~oyl-~-hydroxys~nimi~te (MBS), and disu~inimidylsu~rate (DSS), we were able to produce conjugates of 47 kDa. In one of these (from MBS) both actin and Tfi9 could be identified by imm~oblot~ng. When, in the MBS crosslinking experiments, native actin was replaced with (374-NEM)-actin, the 47 kDa band was not seen, indicating that Cys-374 takes part in the thiol-specific crosslinking reaction. This suggests that part of the binding site of T/39 must be located close to the carboxy-terminus.

No abstract

After polymerization filamentous actin (F-actin) still shows a number of rather narrow 1H NMR signals in its Mg2+ form which are quenched when Mg2+ is replaced by Ca2+. These resonances originate from mobile residues in F-actin. For assignment of these resonances three different strategies were used, the fluorine labeling of Cys-374 by 4-(perfluoro-tert-butyl)phenyliodoacetamide, binding studies with antibodies (Fab) against the seven N-terminal amino acids of actin, and two-dimensional 1H NMR spectroscopy of a highly concentrated F-actin sample. In contrast to the effects detected earlier by 1H NMR spectroscopy, 19F NMR spectroscopy of actin labeled at its C-terminal cysteine shows no significant spectral changes in dependence on the divalent ion present. In its G- (globular) form a strong, narrow 19F resonance can be observed at 15.06 ppm (relative to the external standard trifluoroacetic acid) which is broadened substantially after polymerization of actin. At 283 K the corresponding transverse relaxation time T2 decreases from 16.7 ms to approximately 0.6 ms. These data suggest that the highly mobile residues observed by 1H NMR spectroscopy do not originate from the C-terminus. Binding of Fab directed against the N-terminal amino acids of actin to Mg-F-actin leads to the disappearing of the 1H NMR resonances assigned to a mobile domain in F-actin. This indicates that the mobile region probably comprises the N-terminal amino acids. By homonuclear two-dimensional 1H NMR spectroscopy it was finally possible to sequentially assign the resonances of the mobile domain of F-actin. It turned out that amino acids 1-22 are in a highly mobile state in Mg-F-actin. The nuclear Overhauser effect data indicate that, rather surprisingly, in this high mobility state some of the beta-pleated structure is still conserved. The population of F-actin protomers in the M- (mobile) state can be obtained from the NMR spectra and was determined under different experimental conditions. In the presence of 150 mM KCl approximately half of the protomers in Mg-F-actin are in the M-state. This number is largely independent of the pH in the range studied (pH 7.2-7.8) and of the temperature in range studied (283-310 K). The equilibrium constant KMI for the equilibrium between the I- and M-states is approximately 1.3 under these conditions.

Phallotoxins form tight complexes with filamentous actm and stabilize the polymer against shearing stress. In the present study a phalloidin derivative containing a thiol-capturmg moiety was prepared and reacted with single thiol groups of monomeric muscle actin. Sites of attachment in the protein were Cys-374 next to the C-terminus and Cys-10, close to the N-terminus; the latter was recently shown to be uncovered during a slow but reversible conformational transition occurring in ADP-G-actin. Phalloidin bound to Cys-374 stabilizes filaments agamst shearing stress almost as effectively as free phalloidin, indicating that the phalloidin binding site cannot be far from the C-terminus of actin. Stabilization was also achieved when the phalloidin reagent was added to F-actin, however, the subsequent formation of a covalent linkage with Cys-374 was not observed, most likely due to a restricted mobility of the reactants. In contrast to the efficient stabilization offilaments by phalloidin linked to Cys-374 a destabilizing effect was observed when phalloidin was attached to Cys-10. It appears that phalloidin located close to the N-terminus is unable to bind to the normal binding site in its own filament. Pronounced gelification of this actin derivative suggests that the toxm is able to mediate crosslinking with neighbouring filaments. From these results we conclude that the phallotdin binding site of actin is distant from the N-terminus, but close to the C-terminus. Furthermore, the data provide evidence that binding of phalloidin reduces the mobility of the C-terminus.