Nucleotide states | R_{0,} Å* | Steady state | Lifetime | ||
---|---|---|---|---|---|
FRET efficiency^{†} | r, Å^{‡}(r_{min}–r_{max})^{§} | FRET efficiency^{¶} | r, Å^{‡}(r_{min}–r_{max})^{§} | ||
MV E442A FlAsH: I-actin (Rigor) | 51 | 0.23 ± 0.02 | 62.4 ± 1.2(43.4–81.9) | 0.36 ± 0.05 | 56.1 ± 2.2(40.0–73.7) |
MV E442A FlAsH: I-actin (ATP) | 51 | 0.16 ± 0.01 | 67.2 ± 0.6(46.3–88.3) | 0.24 ± 0.05 | 61.8 ± 3.1(42.6–81.2) |
↵*Förster distance at which the efficiency of energy transfer is 50%.
↵^{†}Steady-state FRET efficiency calculated from the titration curve with I-actin in the presence of ATP (errors represent standard error of the fit). In the rigor state errors represent standard deviation of the mean FRET efficiency.
↵^{‡}Distance (r) between the donor and acceptor probes calculated by using a κ^{2} value of 2/3.
↵^{§}The range of distances (r_{min}–r_{max}) possible based on the maximum and minimum value for κ^{2} determined from the steady-state and limiting anisotropy (20, 21).
↵^{¶}FRET efficiency calculated from the decrease in donor lifetime (E = 1 − τ _{DA}/τ _{D}). The average lifetime 〈τ〉 of the donor was determined in the presence and absence of acceptor. The errors represent the standard deviation of the mean from 3 to 4 separate experiments that included different protein preparations.