Can Förster resonance energy transfer measurements uniquely position troponin residues on the actin filament? A case study in multiple-acceptor FRET.
Straightforward interpretation of Förster resonance energy transfer (FRET) data in terms of the distance from donor-labeled troponon-tropomyosin (TnTm) to acceptor-labeled actin is complicated by the potential for energy transfer to acceptors on neighboring actin monomers (cross-transfer). Calculations indicate that cross-transfer can account for a substantial percentage of the total transfer efficiency. In some cases, this renders isolated FRET data uninterpretable. To overcome these limitations, we have developed an analysis method that incorporates cross-transfer and can, in principle, define the most probable (in the "least-squares" sense) position of a TnTm residue on the actin filament. The technique analyzes data from four or more FRET experiments using acceptors attached to different residues on actin. We have used this method to specify the coordinates of skeletal troponin I (sTnI) residue 133 relative to the actin filament under Mg(2+) and Ca(2+)-saturating conditions. Ca(2+)-activation causes the C terminus of the regulatory domain of TnI to move away from the actin surface by 6.3A, laterally along the actin surface toward actin subdomain 3 by 22.0A, and azimuthally toward the actin inner domain by 13.2A. This information is used to construct a low-resolution structural model of thin filament activation.
Robinson
John M
JM
Department of Biochemistry and Molecular Genetics University of Alabama at Birmingham, 35294-2041, USA. jmr@uab.edu
Dong
Wen-Ji
WJ
Cheung
Herbert C
HC
D000199:Actins / Q000737:chemistry*; D000818:Animals; D002118:Calcium / Q000737:chemistry*; D004735:Energy Transfer; D005456:Fluorescent Dyes; D007700:Kinetics; D008274:Magnesium / Q000737:chemistry*; D008958:Models, Molecular; D018482:Muscle, Skeletal / Q000737:chemistry*; D011487:Protein Conformation; D013050:Spectrometry, Fluorescence; D019209:Troponin C / Q000737:chemistry*; D019210:Troponin I / Q000737:chemistry*
D000199:Actins; D005456:Fluorescent Dyes; D019209:Troponin C; D019210:Troponin I; D008274:Magnesium; D002118:Calcium