Actin plays crucial parts in cell motility through a dynamic process driven by polymerization and depolymerization, that is, the globular (G) to fibrous (F) actin transition. Although our knowledge about the actin-based cellular functions and the molecules that regulate the G- to F-actin transition is growing, the structural aspects of the transition remain enigmatic. We created a model of F-actin using X-ray fibre diffraction intensities obtained from well oriented sols of rabbit skeletal muscle F-actin to 3.3 A in the radial direction and 5.6 A along the equator. Here we show that the G- to F-actin conformational transition is a simple relative rotation of the two major domains by about 20 degrees. As a result of the domain rotation, the actin molecule in the filament is flat. The flat form is essential for the formation of stable, helical F-actin. Our F-actin structure model provides the basis for understanding actin polymerization as well as its molecular interactions with actin-binding proteins.

We have isolated a novel cDNA encoding a peptide with 86% sequence homology to hSNF2L protein, a previously isolated human homologue of Drosophila ISWI. This gene, designated SMARCA5, contained an open reading frame of 3,156 nucleotides encoding a 1,052 amino-acid peptide (hSNF2H). As this product also revealed a significant (73%) identity in amino acid sequence to ISWI, a key component of chromatin-remodeling factors in Drosophila, hSNF2H may be another human homologue of this protein and, as such, could be involved in chromatin remodeling in humans. An ATPase domain characteristic of the SWI2/SNF2 family of proteins was highly conserved in ISWI, hSNF2L, and hSNF2H. Northern-blot analysis demonstrated ubiquitous expression of 5.1-kb and 4.1-kb transcripts of the hSNF2H gene. This gene was mapped by FISH to chromosome bands 4q31.1→q31.2.

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We have used pulsed electron-electron double resonance (PELDOR) spectroscopy to measure the distance between spin labels at Cys 133 of the regulatory region of TnI (TnI133) and a native or genetically substituted cysteine of TnC (TnC44, TnC61, or TnC98). In the ؉Ca 2؉ state, the TnC44-TnI133-T distance was 42 Å , with a narrow distribution (half-width of 9 Å ), suggesting that the regulatory region binds the N-lobe of TnC. Distances for TnC61-TnI133 and TnC98-TnI133 were also determined to be 38 Å (width of 12 Å ) and 22 Å (width of 3.4 Å ), respectively. These values were all consistent with recently pub- Crystal structures of the Tn core domain have been solved for human cardiac (21) and chicken skeletal troponin molecules (22). The Tn core domain has two characteristic subdomains: 1) the "regulatory head," composed of the N-lobe of TnC and the C-terminal region of TnI, and 2) the "I-T arm," composed of long coiled-coil regions from TnT2, TnI, and the C-lobe of TnC. The relative orientation of the regulatory head and the I-T arm may play a fundamental role in the regulatory mechanism of troponin. The optimal docking positions could be ascertained by fitting to a three-dimensional reconstruction from electron microscopic images of thin filaments (23,24). Alternatively, the best orientation or the best spatial relationships could be identified by fitting to polarized fluorescence data from bifunctional rhodamine probes for TnC in muscle fibers (25,26) or fluorescence resonance energy transfer (FRET) between probes attached to various Tn subunits (TnC, TnI, or TnT) on the thin filament (27).Continuous wave electron paramagnetic resonance (CW-EPR) was used for pioneering studies on spin-labeled troponin (28 -30). Recently, we measured CW-EPR spectra from a spin label located on the Cys 133 residue next to the switch segment in the regulatory region of TnI to identify Ca 2ϩ -induced structural states (31) based on the sensitivity of spin-label mobility to flexibility and tertiary contact with a polypeptide (32). The slow spin-label mobility observed for the thin filament in the ϪCa 2ϩ state indicated tertiary contact of Cys 133 with actin because similar slow mobility was found for TnI-actin and TnI-tropomyosin-actin filaments lacking TnC, TnT, or tropomyosin. Furthermore, using nuclear magnetic resonance (NMR) and electron microscopic reconstruction, Murakami et al. (23) determined the structure of the C-terminal portion (131-182) of the regulatory region, including this cysteine, which binds to actin in the ϪCa 2ϩ state.