Table 1.
LRET and distance between the Tb donor-chelate at Trp130 and TMR bound to Cys 108 (gRLC) at 20°C
| S1 Complex Trp130 → Cys108 | Intensity/spectral results (I)
|
Donor lifetime at 490 nm (II)
|
Sensitized emission at 570 nm (III)
|
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|---|---|---|---|---|---|---|---|---|---|
| E.T., % | Distance, Å | Distance change, Å | E.T., % | Distance, Å | Distance change, Å | E.T., % | Distance, Å | Distance change, Å | |
| No ligand (n = 3) | 34.7 ± 4.5 | 66.8 ± 2.3 | N/A | 36.9 ± 4.9 | 65.7 ± 2.3 | N/A | 28.8 ± 5.3 | 69.9 ± 3.1 | N/A |
| MgADP (n = 3) | 35.0 ± 4.1 | 66.6 ± 2.1 | −0.2 ± 0.2 | 37.0 ± 5.8 | 65.7 ± 2.8 | 0.0 ± 0.5 | 28.1 ± 4.9 | 70.3 ± 2.9 | 0.4 ± 0.3 |
| MgADP⋅BeF3(n = 3) | 38.0 ± 4.2 | 65.2 ± 2.0 | −1.6 ± 0.3 | 39.4 ± 6.0 | 64.6 ± 2.7 | −1.2 ± 0.4 | 33.9 ± 5.0 | 67.1 ± 2.6 | −2.8 ± 0.7 |
| MgADP⋅AlF4(n = 3) | 45.0 ± 4.9 | 62.1 ± 2.0 | −4.6 ± 0.2 | 47.4 ± 7.8 | 61.1 ± 3.1 | −4.6 ± 1.1 | 40.4 ± 6.3 | 64.1 ± 2.9 | −5.8 ± 2.6 |
| S1⋅ATP (n = 2) | 41.8 ± 6.3 | 63.5 ± 2.8 | −3.7 ± 0.3 | 44.6 ± 8.4 | 62.3 ± 3.5 | −3.4 ± 0.3 | 33.4 ± 7.0 | 67.5 ± 3.3 | −3.3 ± 0.5 |
| Actin (n = 2) | 32.7 ± 3.6 | 67.7 ± 1.8 | 0.1 ± 0.5 | 35.6 ± 3.6 | 66.3 ± 1.6 | −0.6 ± 0.0 | 27.8 ± 6.0 | 70.5 ± 3.5 | −0.5 ± 0.1 |
| Actin⋅MgADP (n = 2) | 33.2 ± 3.5 | 67.5 ± 1.8 | −0.2 ± 0.5 | 34.4 ± 3.6 | 66.8 ± 1.8 | 0.0 ± 0.1 | 27.3 ± 6.2 | 70.8 ± 3.7 | −0.2 ± 0.0 |
The distances calculated are from three different methods: intensity (spectral) measurements, lifetime measurements of the donor, and lifetime measurements of acceptor’s sensitized emission. They all produce the same trends: namely, that actin has little effect on the distance and that nucleotide-phosphate analogs decrease this distance to varying degrees. The “n” value refers to the number of separate protein preparations, with each protein preparation having several (>3) measurements. The uncertainties are mean ± SD. The changes in distance compared to S1 without exogenous nucleotides are calculated on a pair-wise basis (same protein preparation ± ligands), and, hence, the uncertainties are much smaller than the absolute distances, in which the main source of random error is the exact amount of acceptor exchange. N/A, not applicable.