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The soluble, globular, α-helix-rich peptide SipA446–684 is a domain of a bacterial protein that binds to mammalian filamentous-actin and re-arranges the host cell's cytoskeleton. We show that adding two copies of NHBP-1, a carbon nanomaterial binding peptide, to its N-terminal can induce SipA446–684 to polymerize and assume a fibrillar structure under physiological conditions. The fibrils formed showed thioflavine T and Congo red staining profiles that are characteristic of and specific for amyloid-like structures. The α-helical structure of the globular protein was retained in the fibrils, suggesting the appended NHBP-1 sequence plays a key role in the formation of cross-β spines within the fibrils. Consistent with that idea, we observed that a synthetic NHBP-1 peptide can form an amyloid-like structure under appropriate conditions. Thus, our findings add a new subtype of amyloid-like structure formation and suggest this method of assembly could be exploited in nano-biotechnology. [ABSTRACT FROM PUBLISHER]

Fluorescence resonance energy transfer between points on tropomyosin (positions 87 and 190) and actin (Gln-41, Lys-61, Cys-374, and the ATP-binding site) showed no positional change of tropomyosin relative to actin on the thin filament in response to changes in Ca2+ concentration (Miki et al. (1998) J. Biochem. 123, 1104–1111). This is consistent with recent electron cryo-microscopy analysis, which showed that the C-terminal one-third of tropomyosin shifted significantly towards the outer domain of actin, while the N-terminal half of tropomyosin shifted only a little (Narita et al. (2001) J. Mol. Biol. 308, 241–261). In order to detect any significant positional change of the C-terminal region of tropomyosin relative to actin, we generated mutant tropomyosin molecules with a unique cysteine residue at position 237, 245, 247, or 252 in the C-terminal region. The energy donor probe was attached to these positions on tropomyosin and the acceptor probe was attached to Cys-374 or Gln-41 of actin. These probe-labeled mutant tropomyosin molecules retain the ability to regulate the acto-S1 ATPase activity in conjunction with troponin and Ca2+. Fluorescence resonance energy transfer between these points of tropomyosin and actin showed a high transfer efficiency, which should be very sensitive to changes in distance between probes attached to actin and tropomyosin. However, the transfer efficiency did not change appreciably upon removal of Ca2+ ions, suggesting that the C-terminal region of tropomyosin did not shift significantly relative to actin on the reconstituted thin filament in response to the change of Ca2+ concentration. [ABSTRACT FROM PUBLISHER]

Fluorescence resonance energy transfer (FRET) spectroscopy has been used to determine spatial relationships between residues on tropomyosin and actin in reconstituted muscle thin filament, and to detect a positional change of tropomyosin relative to actin on the thin filament in the presence and absence of Ca2+ ions. In addition to Cys-190 which is a single cysteine residue in rabbit skeletal muscle α-tropomyosin, a new site, Cys-87 which is a unique cysteine residue in a mutant α-tropomyosin, was labeled with a resonance energy donor molecule, 5-(2-iodoacetylaminoethyl)aminonaphthalene 1-sulfonic acid (IAED-ANS). On the other hand, Gln-41, Lys-61, Cys-374, and the ATP-binding site of actin were selectively labeled with acceptor probes: fluorescein cadaverine, fluorescein 5-isothiocyanate, 4-dimethyl-aminophenylazophenyl 4'-maleimide, and TNP-ATP (or TNP-ADP), respectively. The distances between probes attached to position 87 of the mutant tropomyosin and Gln-41, Lys-61, Cys-374, or the nucleotide-binding site of actin on the reconstituted thin filament in the presence of Ca2+ ion were measured to be 43.2, 49.7, 45.4, and 35.2 A, respectively, and the distance between probes attached to position 190 of tropomyosin and Gln-41 or the nucleotide-binding site of actin were 51.6 and 43.1 Å, respectively. The transfer efficiencies between these donor and acceptor molecules were large, so that the efficiency should be very sensitive to changes in distance between probes attached to tropomyosin and actin. However, the transfer efficiency did not change appreciably upon removal of Ca2+ ions, suggesting that tropomyosin does not change its position on the reconstituted thin filament in response to a change in Ca2+ ion concentration. The present results do not support the notion of tropomyosin movement on skeletal muscle thin filaments as proposed in the steric blocking theory. [ABSTRACT FROM AUTHOR]