György Hegyi

Studies on the properties of chemically modified actin III. Carbethoxylation

Abstract Diethylpyrocarbonate was used for the specific carbethoxylation of the histidyl residues of G- and F-actins. The characteristic properties of carbethoxylated actins were studied. The extent of carbethoxylation depended on the actin to reagent ratio, and equal numbers of histidyl residues were carbethoxylated in G- and F-actins. The number of tyrosine and SH groups did not change on diethylpyrocarbonate treatment. 30% of the bound nucleotide of G-actin was removed, whereas a high degree of carbethoxylation did not change the bound nucleotide of F-actin. The characteristic properties of F-actin (viscosity, repolymerizability, actomyosin formation, activation of myosin ATPase) were not altered by the treatment even on carbethoxylation of 70% of the histidyl residues. The characteristic properties of G-actin (polymerizability, actomyosin formation and activation of myosin ATPase) were almost lost on carbethoxylation of about half the histidyl residues. The treatment did not change the helix content of G-actin (the 233-mμ Cotton effect). These results lead to the assumption that the histidyl residues, which have some role in determining the characteristic properties of actin, are buried in F-actin.

para-Nitroblebbistatin, the non-cytotoxic and photostable myosin II inhibitor.

Blebbistatin, the best characterized myosin II-inhibitor, is commonly used to study the biological roles of various myosin II isoforms. Despite its popularity, the use of blebbistatin is greatly hindered by its blue-light sensitivity, resulting in phototoxicity and photoconversion of the molecule. Additionally, blebbistatin has serious cytotoxic side effects even in the absence of irradiation, which may easily lead to the misinterpretation of experimental results since the cytotoxicity-derived phenotype could be attributed to the inhibition of the myosin II function. Here we report the synthesis as well as the in vitro and in vivo characterization of a photostable, C15 nitro derivative of blebbistatin with unaffected myosin II inhibitory properties. Importantly, para-nitroblebbistatin is neither phototoxic nor cytotoxic, as shown by cellular and animal tests; therefore it can serve as an unrestricted and complete replacement of blebbistatin both in vitro and in vivo.

Effect of intramolecular cross-linking between glutamine-41 and lysine-50 on actin structure and function.

Subdomain 2 of actin is a dynamic segment of the molecule. The cross-linking of Gln-41 on subdomain 2 to Cys-374 on an adjacent monomer in F-actin inhibits actomyosin motility and force generation (Kim et al., 1998; Biochemistry 37, 17,801–17,809). To shed light on this effect, additional modifications of the Gln-41 site on actin were carried out. Both intact G-actin and G-actin cleaved by subtilisin between Met-47 and Gly-48 in the DNase 1 binding loop of subdomain 2 were treated with bacterial transglutaminase. According to the results of Edman degradation, transglutaminase introduced an intramolecular zero-length cross-linking between Gln-41 and Lys-50 in both intact and subtilisin cleaved actins. This cross-linking perturbs G-actin structure as shown by the inhibition of subtilisin and tryptic cleavage in subdomain 2, an allosteric inhibition of tryptic cleavage at the C-terminus and decrease of modification rate of Cys-374. The cross-linking increases while the subtilisin cleavage dramatically decreases the thermostability of F-actin. The Mg- and S1-induced polymerizations of both intact and subtilisin cleaved actins were only slightly influenced by the cross-linking. The activation of S1 ATPase by actin and the sliding speeds of actin filaments in the in vitro motility assays were essentially unchanged by the cross-linking. Thus, although intramolecular cross-linking between Gln-41 and Lys-50 perturbs the structure of the actin monomer, it has only a small effect on actin polymerization and its interaction with myosin. These results suggest that the new cross-linking does not alter the intermonomer interface in F-actin and that changes in actomyosin motility reported for the Gln-41–Cys-374 intrastrand cross-linked actin are not due to decreased flexibility of loop 38–52 but to constrains introduced into the F-actin structure and/or to perturbations at the actins C-terminus.

Azidoblebbistatin, a photoreactive myosin inhibitor

Photoreactive compounds are important tools in life sciences that allow precisely timed covalent crosslinking of ligands and targets. Using a unique technique we have synthesized azidoblebbistatin, which is a derivative of blebbistatin, the most widely used myosin inhibitor. Without UV irradiation azidoblebbistatin exhibits identical inhibitory properties to those of blebbistatin. Using UV irradiation, azidoblebbistatin can be covalently crosslinked to myosin, which greatly enhances its in vitro and in vivo effectiveness. Photo-crosslinking also eliminates limitations associated with the relatively low myosin affinity and water solubility of blebbistatin. The wavelength used for photo-crosslinking is not toxic for cells and tissues, which confers a great advantage in in vivo tests. Because the crosslink results in an irreversible association of the inhibitor to myosin and the irradiation eliminates the residual activity of unbound inhibitor molecules, azidoblebbistatin has a great potential to become a highly effective tool in both structural studies of actomyosin contractility and the investigation of cellular and physiological functions of myosin II. We used azidoblebbistatin to identify previously unknown low-affinity targets of the inhibitor (EC50 ≥ 50 μM) in Dictyostelium discoideum, while the strongest interactant was found to be myosin II (EC50 = 5 μM). Our results demonstrate that azidoblebbistatin, and potentially other azidated drugs, can become highly useful tools for the identification of strong- and weak-binding cellular targets and the determination of the apparent binding affinities in in vivo conditions.

A highly soluble, non-phototoxic, non-fluorescent blebbistatin derivative

Blebbistatin is a commonly used molecular tool for the specific inhibition of various myosin II isoforms both in vitro and in vivo. Despite its popularity, the use of blebbistatin is hindered by its poor water-solubility (below 10 micromolar in aqueous buffer) and blue-light sensitivity, resulting in the photoconversion of the molecule, causing severe cellular phototoxicity in addition to its cytotoxicity. Furthermore, blebbistatin forms insoluble aggregates in water-based media above 10 micromolar with extremely high fluorescence and also high adherence to different types of surfaces, which biases its experimental usage. Here, we report a highly soluble (440 micromolar in aqueous buffer), non-fluorescent and photostable C15 amino-substituted derivative of blebbistatin, called para-aminoblebbistatin. Importantly, it is neither photo- nor cytotoxic, as demonstrated on HeLa cells and zebrafish embryos. Additionally, para-aminoblebbistatin bears similar myosin II inhibitory properties to blebbistatin or para-nitroblebbistatin (not to be confused with the C7 substituted nitroblebbistatin), tested on rabbit skeletal muscle myosin S1 and on M2 and HeLa cells. Due to its drastically improved solubility and photochemical feature, as well as lack of photo- or cytotoxicity, para-aminoblebbistatin may become a feasible replacement for blebbistatin, especially at applications when high concentrations of the inhibitor or blue light irradiation is required.

Intermonomer cross-linking of F-actin alters the dynamics of its interaction with H-meromyosin in the weak-binding state.

Previous cross‐linking studies [Kim E, Bobkova E, Hegyi G, Muhlrad A & Reisler E (2002) Biochemistry41, 86–93] have shown that site‐specific cross‐linking among F‐actin monomers inhibits the motion and force generation of actomyosin. However, it does not change the steady‐state ATPase parameters of actomyosin. These apparently contradictory findings have been attributed to the uncoupling of force generation from other processes of actomyosin interaction as a consequence of reduced flexibility at the interface between actin subdomains‐1 and ‐2. In this study, we use EPR spectroscopy to investigate the effects of cross‐linking constituent monomers upon the molecular dynamics of the F‐actin complex. We show that cross‐linking reduces the rotational mobility of an attached probe. It is consistent with the filaments becoming more rigid. Addition of heavy meromyosin (HMM) to the cross‐linked filaments further restricts the rotational mobility of the probe. The effect of HMM on the actin filaments is highly cooperative: even a 1 : 10 molar ratio of HMM to actin strongly restricts the dynamics of the filaments. More interesting results are obtained when nucleotides are also added. In the presence of HMM and ADP, similar strongly reduced mobility of the probe was found than in a rigor state. In the presence of adenosine 5′[βγ‐imido] triphosphate (AMPPNP), a nonhydrolyzable analogue of ATP, weak binding of HMM to either cross‐linked or native F‐actin increases probe mobility. By contrast, weak binding by the HMM/ADP/AlF4 complex has different effects upon the two systems. This protein–nucleotide complex increases probe mobility in native actin filaments, as does HMM + AMPPNP. However, its addition to cross‐linked filaments leaves probe mobility as constrained as in the rigor state. These findings suggest that the dynamic change upon weak binding by HMM/ADP/AlF4 which is inhibited by cross‐linking is essential to the proper mechanical behaviour of the filaments during movement.

The role of Mg2+ in the contraction and adenosine triphosphatase activity of myofibrils

Abstract 1. 1. The activation by Mg2+ of the ATP splitting of myofibrils depends on the concentration of Ca2+ ions present. If the concentration of Ca2+ is high enough to suppress the dissociation of actomyosin, Mg2+ activates, otherwise it inhibits. 2. 2. The course of the curve describing the activation by Mg2+ does not depend on the concentration of ATP present. 3. 3. In the presence of 10−4 M Ca2+, the addition of increasing concentrations of Mn2+, Ba2+, Sr2+ or Ca2+ causes an activation much resembling that caused by Mg2+. Activation is lowest when Ca2+ is the only bivalent cation present. 4. 4. Mg2+ and Ca2+ are both essential for contraction. In the presence of 2·10−5 M Mg2+ and 2·10−3 M ATP there is no contraction unless Ca2+ in a concentration sufficient to repress dissociation (approx. 10−6 M) is also present. In the presence of 10−5 M Ca2+ a maximal rate of contraction is reached only when a minimal level of Mg2+ concentration (approx. 10−6 M) is reached. There is, however, contraction in a Ca2+-free milieu too if the concentration of MgATP does not exceed 1·10−5 M. 5. 5. The substrate proper of myofibrillar ATPase (ATP phosphohydrolase, EC 3.6.1.3) is shown to be free ATP. The inhibition by a total Mg2+ concentration exceeding that of ATP is thus caused by the decrease in available substrate, i.e. free ATP. If a constant free ATP concentration is ensured there is no inhibition even by 10−2 M Mg2+.