Residues 262-274 form a loop between subdomains 3 and 4 of actin. This loop may play an important role in actin filament formation and stabilization. To assess directly the behavior of this loop, we mutated Ser 265 of yeast actin to cysteine (S265C) and created another mutant (S265C/C374A) by changing Cys 374 of S265C actin to alanine. These changes allowed us to attach a pyrene maleimide stoichiometrically to either Cys 374 or Cys 265 . These mutations had no detectable effects on the protease susceptibility, intrinsic ATPase activity, and thermal stability of labeled or unlabeled G-actin. The presence of the loop cysteine, either labeled or unlabeled, did not affect the actin-activated S1 ATPase activity or the in vitro motility of the actin. Both mutant actins, either labeled or unlabeled, nucleated filament formation considerably faster than wild-type (WT) actin, although the critical concentration was not affected. Whereas the fluorescence of the C-terminal (WT) probe increased during polymerization, that of the loop (S265C/C374A) probe decreased, and the fluorescence of the doubly labeled actin (S265C) was ϳ50% less than the sum of the fluorescence of the individual fluorophores. Quenching was also observed in copolymers of labeled WT and S265C/C374A actins. An excimer peak was present in the emission spectrum of labeled S265C F-actin and in the labeled S265C/C374A-WT actin copolymers. These results show that in the filaments, the C-terminal pyrene of a substantial fraction of monomers directly interacts with the loop pyrene of neighboring monomers, bringing the two cysteine sulfurs to within 18 Å of one another. Finally, when bound to labeled S265C/ C374A F-actin, myosin S1, but not tropomyosin, caused an increase in fluorescence of the loop probe. Both proteins had no effect on excimer fluorescence. These results help establish the orientation of monomers in Factin and show that the binding of S1 to actin subdomains 1 and 2 affects the environment of the loop between subdomains 3 and 4.The crystal structure of the actin monomer has been elucidated as part of a 1:1 complex with three actin-binding proteins (1-3). However, the structure of the two-stranded actin filament at atomic resolution has not been determined due to the inability to date to crystallize F-actin. One F-actin model, proposed by Schutt et al. (2,4), is based on the profilin/␤-actin ribbon structure in which actin monomers contact each other in a continuous fashion, with profilin molecules bridging between the actins on the outside of the structure. In this structure, actin subdomains 1 and 2 are near the center of the ribbon, whereas the subdomain 3/4 interface is near its exterior. Schutt et al. have proposed that the ribbon can be transformed into a classical ADP-containing helical filament by a compression and a twist. However, coordinates of this filament model have not yet been published.Holmes et al. (5) have generated an alternative model based on fitting the coordinates of the monomer into a density map generated from low angle x...

Holmes proposed that in F-actin, hydrophobic residues in a subdomain 3/4 loop interact with a hydrophobic pocket on the opposing strand resulting in helix stabilization. We have determined how a decreased hydrophobicity of this plug affects yeast actin function. Cells harboring only the V266G, V266D, V266F, L267G, L269D, or L269K actins appear normal, although V266G cells display an altered budding pattern. However, V266G,L267G (GG) double mutant cells are cold-sensitive with randomly oriented thick actin assemblies seen in rhodamine phalloidin-stained GG cells. V266D actin polymerizes slower than wild-type actin at room temperature. At 4°C, not only is polymerization slowed, but there is also an effect on critical concentration. However, the polymerization defects are milder than those associated with substitution of Asp for the neighboring Leu 267. Purified GG-actin does not polymerize in vitro alone or in the presence of wildtype F-actin seeds. GG-actin polymerization can be restored by larger amounts of wild-type actin, beryllium fluoride, or phalloidin at room temperature, although at 4°C only phalloidin is effective. These results suggest that the diminished hydrophobicity of the plug in GG-actin leads to filament destabilization. However, the V266D actin results require a modification of the original Holmes filament model.

A major function of tropomyosin (TPM) in nonmuscle cells may be stabilization of F-actin by binding longitudinally along the actin filament axis. However, no clear evidence exists in vitro that TPM can significantly affect the critical concentration of actin. We previously made a polymerization-defective mutant actin, GG (V266G, L267G). This actin will not polymerize alone at 25°C but will in the presence of phalloidin or beryllium fluoride. With beryllium fluoride, but not phalloidin, this polymerization rescue is cold-sensitive. We show here that GG-actin polymerizability was restored by cardiac tropomyosin and yeast TPM1 and TPM2 at 25°C with rescue efficiency inversely proportional to TPM length (TPM2 > TPM1 > cardiac tropomyosin), indicating the importance of the ends in polymerization rescue. In the presence of TPM, the apparent critical concentration of actin is 5.5 M, 10-15-fold higher than that of wild type actin but well below that of the GG-actin alone (>20 M). Non N-acetylated TPMs did not rescue GGactin polymerization. The TPMs did not prevent cold-induced depolymerization of GG F-actin. TPM-dependent GG-actin polymerization did not occur at temperatures below 20°C. Polymerization rescue may depend initially on the capture of unstable GG-F-actin oligomers by the TPM, resulting in the strengthening of actin monomer-monomer contacts along the filament axis.Tropomyosins are a family of highly conserved eukaryotic actin-binding proteins with molecular masses ranging from 19 kDa for yeast TPM2 1 (1) to 40 kDa for rat fibroblast TPM1 (2). Tropomyosins possess a dimeric ␣-helical coiled-coil structure along virtually the entire length of the protein, and heptad repeat motifs with hydrophobic residues at positions 1 and 4 are found along the entire molecule. These quasirepeat regions appear to match up with actin monomers when tropomyosin binds to F-actin along the filament axis and may serve as weak actin binding regions. Tropomyosins have a basic amino terminus, which is usually N-acetylated, and for most tropomyosins, the acetyl group is believed to be important for preservation of the helical structure at the end of the protein (3). Adjacent tropomyosins form head-to-tail overlapping interactions along the length of the actin filament (4), and these overlaps are believed to be the major site of interaction between F-actin and tropomyosin (5, 6). Recombinant skeletal muscle tropomyosin with either an unacetylated N terminus (7) or with a C-terminal deletion (5) loses the ability to bind to actin. The interaction between actin and the internal quasirepeat regions of tropomyosin is believed to be weaker but still critical for cooperative binding to F-actin actin (8), and there is a hierarchy of importance among these quasirepeat regions (9).The role of tropomyosin, along with troponin, as a mediator of calcium regulation of thin filament function in sarcomeric muscle is well established. However, its role in nonmuscle cells, where there is no troponin, is poorly understood. Based on its ability to interact...

The hydrophobicity of the subdomain 3/4 hydrophobic loop (262-274) has been implicated to be essential for actin's function. We previously showed (Kuang, B., and Rubenstein, P. A. (1997) J. Biol. Chem. 272, 1237-1247) that a mutant yeast actin (V266G/L267G) with markedly decreased hydrophobicity in this loop conferred severe cold sensitivity to its polymerization. Here we further tested the mutational effect on the conformation and function of G-actin. This GG mutation caused no significant changes in overall secondary structure or in the microenvironment around actin's tryptophan residues, nor did it alter the dissociation constant of G-actin for ATP. However, it lowers the intrinsic ATPase activity and the melting temperature for Mg-GG actin from 51 to 33°C and transforms the conformation of subdomain 2 and the central cleft of G-actin into an F-monomer-like structure. The results suggest that the hydrophobic plug may not only play a role in actin filament stabilization but also may be important for controlling the stability of G-actin and for promoting the conformational change of the monomer needed for addition to a growing actin filament.

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