Daniela Heintz

Use of bimanyl actin derivative (TMB-actin) for studying complexation of β-thymosins: Inhibition of actin polymerization by thymosin β9

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 thymosin β4 (Tβ4), 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 K d value of the thymosin β4 complex as 0.8 μM, a value that is in agreement with previous determinations. In addition to the main compound thymosin β4, bovine tissue contains a related peptide, thymosin β9 (Tβ9), which has 41 amino acid residues and ca. 75% sequence homology. In the present study we show for the first time that Tβ9, similar to Tβ4, forms a 1:1 complex with monomeric actin, and hereby inhibits actin polymerization. With a K d value of 1.1 μM the affinity of Tβ9 is in the same range as that of Tβ4, suggesting that Tβ9, like Tβ4, contributes to maintaining the pool of monomeric actin in bovine non‐muscle cells. Further proof of the interaction of Tβ9 with actin was provided by native PAGE, where the complex showed the reported higher mobility, as well as by crosslinking experiments. Using different crosslinking reagents, like water‐soluble carbodiimide (EDC), m‐maleimidobenzoyl‐N‐hydroxysuccinimidate (MBS), and disuccinimidylsuberate (DSS), we were able to produce conjugates of 47 kDa. In one of these (from MBS) both actin and Tβ9 could be identified by immunoblotting. 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 Tβ9 must be located close to the carboxy‐tenninus.

Probing the phalloidin binding site of actin

Phallotoxins form tight complexes with filamentous actin and stabilize the polymer against shearing stress. In the present study a phalloidin derivative containing a thiol‐capturing 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 against 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 of filaments 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 toxin is able to mediate crosslinking with neighbouring filaments. From these results we conclude that the phalloidin 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.

Reversible introduction of thiol compounds into proteins by use of activated mixed disulfides.

Publisher Summary This chapter discusses the reversible introduction of thiol compounds into proteins by use of activated mixed disulfides. For the preparation of synthetic mixed disulfides, equilibrium reactions are unsuitable. Instead, the thiol groups of the protein are subjected to an activation reaction yielding an activated mixed disulfide (AMD) of the protein. Because of favorable equilibrium constants, AMDs require only 1-3 equivalents of the thiol compound to be reacted. The yellow-colored thionitrobenzoate anion (TNB) anion is a convenient tool for monitoring the kinetics of the S -alkylthiolation reaction at 412 nm. For spectrophotometric control, however, the protein AMD must be separated from excess Ellmans reagent and the TNB formed during the preparation—for example, by gel filtration. The derivatization of protein thiols via activated mixed disulfides has several advantages over other derivatization procedures. First, the reaction kinetics can be followed spectrophotometrically. Second, the procedure allows introducing arbitrary residues into proteins almost independent of molecular weight, provided they are available as thiol compounds. This makes thiol-specific derivatization of proteins independent of the availability of the corresponding maleimide or alkyl iodide derivatives. In addition, residues introduced this way into a protein are linked by disulfide bridges, which can be easily cleaved under physiological conditions.

Identification of contact sites in the actin-thymosin beta 4 complex by distance-dependent thiol cross-linking.

Binding sites of actin and thymosin β4 were investigated using a set of bifunctional thiol-specific reagents, which allowed the insertion of cross-linkers of defined lengths between cysteine residues of the complexed proteins. After the cross-linkers were attached to actin specifically at either Cys, Cys, or to the sulfur atom of the ATP analog adenosine 5′-O-(thiotriphosphate) (ATPS), the actin derivatives were reacted with synthetic thymosin β4 analogs containing a cysteine at one of the positions 6, 17, 28, 34, and 40. Immediate cross-linking as followed by UV spectroscopy was found for Cys of actin and Cys6 of thymosin β4, indicating that the N terminus of thymosin β4 is in close proximity (≤9.2 Å) to the C terminus of actin. In contrast, only insignificant reactivity was measured for all thymosin β4 analogs when the cross-linkers were anchored at Cys of actin. A second contact site was identified by cross-linking of Cys and Cys in thymosin β4 with the ATPS derivative bound to actin, indicating that the hexamotif of thymosin β4 (positions 17-22) is in close proximity (≤9.2 Å) to the nucleotide. The importance of the amino acids 17 and 28 in thymosin β4 for the interaction with actin was emphasized by the finding that thymosin analogs containing cysteine in these positions exhibited strongly reduced abilities to inhibit actin polymerization.

The ternary complex of DNase I, actin and thymosin β4

We have recently described a method for identifying contact sites between actin and thymosin β4 (Tβ4) by following spectrophotometrically the extent and kinetics of distinct, thiolspecific crosslinking reactions between appropriate derivatives of the two proteins [Reichert et al. (1996) J. Biol. Chem. 271, 1301–1308]. In the present study this method was used to show that such crosslinking, which is indicative of complex formation, occurs to the same extent with the actin‐DNase I complex as with pure actin, although at a somewhat lower rate. Further evidence for the formation of the ternary complex was given by gel electrophoresis. From fluorescence spectroscopy the K D value of Tβ4 from the actin‐DNase I complex was found to be identical to that from pure actin. In line with these data, the capacity of actin for inhibiting DNase I was not affected by the addition of Tβ4. In conclusion, DNase I and Tβ4 are independent of each other in their interaction with actin, suggesting that the binding sites of thymosin β4 and DNase I on actin do not overlap. A ternary complex of DNase I, actin and Tβ4, if obtained in crystalline form, could thus provide an approach for studying the interface of Tβ4 and actin by X‐ray analysis.

Interchain and intrachain crosslinking of actin thiols by a bifunctional thiol reagent

From 1,9‐nonylenedithiol and Ellmans Reagent the bifunctional asymmetric disulfide n‐nonylene‐1,9‐bis‐[5‐dithio‐(2‐nitrobenzoic acid)] (NBDN) was prepared. By monovalent reaction with cysteine‐374 the crosslinker could be introduced into monomeric actin, with release of one equivalent of yellow 2‐nitro‐5‐thiobenzoate (NTB). From the monovalent actin derivative we prepared a crosslinked actin dimer (Cys‐374–Cys‐374′) as well as a monomer with a crosslink between Cys‐374 and Cys‐10. Neither crosslinked actin species was able to polymerize the crosslinked monomer even in the presence of phalloidin. The crosslinked monomer polymerized on the addition of dithiothreitol, thus providing the first unpolymerizable actin species whose polymerizability can be restored under mild conditions. We suggest the use of NBDN as a thiol‐specific crosslinker that reacts under spectrophotometric control and can be removed by the addition of thiols.

Polymerization of actin from the thymosin β4 complex initiated by the addition of actin nuclei, nuclei stabilizing agents or myosin S1

Thymosin β4 forms a 1:1 complex with actin and thereby prevents polymerization. Rapid formation of filaments from this complex was observed, however, when actin trimers were added. Polymerization can likewise be initiated by the addition of one equivalent of phalloidin or, less effectively, cytochalasin B. Since both toxins, which reportedly support nucleation, have similar effects as the covalently linked actin trimers, it appears that the formation of filaments from the actin—thymosin β4 complex depends on the availability of stable actin nuclei. Remarkably, rapid polymerization was also observed if small amounts of myosin S1 were added, suggesting that also myosin, a protein functionally connected with polymeric actin, can serve as a nucleation center. Considering the existence of thymosin β4 and related peptides in numerous mammalian tissues, our data suggest that spontaneous formation of microfilaments in non‐muscle cells may be regulated at the level of nucleation. Uncontrolled polymerization induced by the formation of phalloidin‐stabilized nuclei may explain the acute toxic effects of phalloidin in hepatocytes.

Mobile segments in rabbit skeletal muscle F−actin detected by 1H nuclear magnetic resonance spectroscopy

Polymerization of actin by increasing the ionic strength leads to a quenching of almost all 1H NMR signals. Surprisingly, distinct signals with relatively small line widths can still be observed in actin filaments (F‐actin) indicating the existence of mobile, NMR visible residues in the macromolecular structure. The intensity of the F‐actin spectrum is much reduced if one replaces Mg2+ with Ca2+, and a moderate reduction of the signal intensity can also be obtained by increasing the ionic strength. These results can be explained in a two‐state model of the actin protomers with a M‐ (mobile) state and a I‐ (immobile) state in equilibrium. In the M‐state a number of residues in the actin protomer are mobile and give rise to observable NMR signals. This equilibrium is shifted towards the I‐state specifically by replacing Mg2+ with Ca2+‐ions and unspecifically by addition of monovalent ions such as K+. The binding of phalloidin to its high‐affinity site in the filaments does not influence the equilibrium between M‐ and I‐state. Phalloidin itself is completely immobilized in F‐actin, its exchange with the solvent being slow on the NMR time scale.