Nanosecond fluorescence depolarization studies on actin labeled with 1,5‐IAEDANS and dansyl chloride

The interaction of the alphaalpha, betabeta, and alphabeta smooth muscle tropomyosin (Tm) isoforms with F-actin was systematically studied in the absence and in the presence of myosin subfragment 1 (S1) using multifrequency phase/modulation Förster resonance energy transfer (FRET). A Gaussian double distance distribution model was adopted to fit FRET data between a 5-(2-iodoacetyl-amino-ethyl-amino)naphthalene-1-sulfonic acid donor at either Cys-36 of the beta-chain or Cys-190 of the alpha-chain and a 4-dimethylaminophenylazophenyl 4'-maleimide acceptor at Cys-374 of F-actin. Experimental data were obtained for singly and doubly labeled alphabeta Tm (donor only at alpha, only at beta, or both) and for doubly labeled alphaalpha or betabeta Tm. Data for singly labeled alphabetaTm were combined in a global analysis with doubly labeled alphabetaTm. In all doubly labeled isoforms, upon S1 binding, one donor-acceptor "apparent" distance increased slightly by 0.5-2 A, whereas the other decreased by 6-9 A. These changes are consistent with a uniform "rolling" motion of Tm over the F-actin surface. The analysis indicates that Tm occupies relatively well-defined positions, with some flexibility, in both the predominantly closed (-S1) and open (+S1) thin-filament states. The results for the alphabetaTm heterodimer indicate that the local twofold symmetry of alphaalpha or betabeta Tm is effectively broken in alphabetaTm bound to F-actin, which implies a difference between the alpha- and beta-chains in terms of their interaction with F-actin.

Section: Distance Distribution Model Analysismentioning

confidence: 99%

Myosin-Induced Movement of αα, αβ, and ββ Smooth Muscle Tropomyosin on Actin Observed by Multisite FRET

Bacchiocchi¹,

Graceffa

Lehrer

2004

“…16) was 2 ⁄3, the value averaged over a fast-reorienting and random distribution of label orientations. Discussions on the validity of this approximation, on the basis of substantial rapid reorientation of both donor and acceptor on the fluorescence time scale, have been presented: for Tm donor-labeled with AEDANS at Cys-190 (Tao et al, 1983), for F-actin acceptor-labeled with DABMI at Cys-374 (Tao, 1978), and for F-actin acceptor-labeled with TRITC-Ph (Heidecker et al, 1995). The good agreement between the modeled and the measured inter-chain distances on Tm, presented in the previous section, is a further confirmation of the correctness of this approximation.…”

Section: Orientation Factormentioning

confidence: 99%

Ca2+-Induced Movement of Tropomyosin in Skeletal Muscle Thin Filaments Observed by Multi-Site FRET

Bacchiocchi¹,

Lehrer

2002

To obtain information on Ca(2+)-induced tropomyosin (Tm) movement in Ca(2+)-regulated muscle thin filaments, frequency-domain fluorescence energy transfer data were collected between 5-(2-iodoacetyl-amino-ethyl-amino)naphthalene-1-sulfonic acid at Cys-190 of Tm and phalloidin-tetramethylrhodamine B isothiocyanate bound to F-actin. Two models were used to fit the experimental data: an atomic coordinate (AC) model coupled with a search algorithm that varies the position and orientation of Tm on F-actin, and a double Gaussian distance distribution (DD) model. The AC model showed that little or no change in transfer efficiency is to be expected between different sites on F-actin and Tm if Ca(2+) causes azimuthal movement of Tm of the magnitude suggested by structural data (C. Xu, R. Craig, L. Tobacman, R. Horowitz, and W. Lehman. 1999. Biophys. J. 77:985-992). However, Ca(2+) produced a small but significant change in our phase/modulation versus frequency data, showing that changes in lifetime decay can be detected even when a change of the steady-state transfer efficiency is very small. A change in Tm azimuthal position of 17 on the actin filament obtained with the AC model indicates that solution data are in reasonable agreement with EM image reconstruction data. In addition, the data indicate that Tm also appears to rotate about its axis, resulting in a rolling motion over the F-actin surface. The DD model showed that the distance from one of the two chains of Tm to F-actin was mainly affected, further verifying that Ca(2+) causes Tm to roll over the F-actin surface. The width of the distance distributions indicated that the position of Tm in absence and in presence of Ca(2+) is well defined with appreciable local flexibility.

“…r 2 /N is then minimized using X, Y, and Z as variable parameters. Fluorescence anisotropy decay was measured according to methods described in Tao (1978). The anisotropy A(t) was calculated using the equation:…”

Section: Fluorescence Measurements and Data Analysismentioning

confidence: 99%

Proximity Relationships between Residue 117 of Rabbit Skeletal Troponin-I and Residues in Troponin-C and Actin

Gergely

Tao

2001

Self Cite

We used resonance energy transfer and site-directed photo-cross-linking to probe the Ca(2+)-dependent proximity relationships between residue 117 next to the C-terminus of the inhibitory region in rabbit skeletal troponin-I (TnI) and residues in troponin-C (TnC) and in actin. A mutant TnI that contains a single cysteine at position 117 (I117) was constructed, and the distance between TnI residue 117 and TnC residue 98 was measured with the following results: for both the binary TnC-TnI complex and the ternary troponin complex, this distance was 30 and 41 A in the presence and absence of Ca(2+), respectively. The distance between TnI residue 117 and Cys374 of actin was 48 and 41 A in the presence and absence of Ca(2+), respectively. Six additional distances from this TnI residue to cysteines in TnC mutants were measured and used to localize this residue with respect to the crystal structure of TnC. The results show that in the presence of Ca(2+) it is localized near the B and C helices of TnC's N-terminal domain. In the absence of Ca(2+) this residue moves away from this location by approximately 8 A. Photo-cross-linking experiments show that I117 labeled with 4-maleimidobenzophenone photo-cross-linked to TnC but not to actin in both the presence and absence of Ca(2+). Taken together these results provide independent experimental support for the proposal (Y. Luo, J. L. Wu, B. Li, K. Langsetmo, J. Gergely, and T. Tao, 2000, J. Mol. Biol. 296:899-910) that upon Ca(2+) removal the region comprising TnI residues 114-125 triggers the movements of residues 89-113 and 130-150 toward actin, but does not itself interact with actin.