Fluorescence studies of 1,N6-ethenoadenosine triphosphate bound to G-actin: The nucleotide base is inaccessible to water

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Abstract

When 1,N6-ethenoadenosine triphosphate (ε-ATP) is free in solution, its fluorescence is collisionally quenched by iodide ion, by methionine, by tryptophan, and by cysteine. None of these quenches the fluorescence of ε-ATP bound to G-actin. Thus, the ethenoadenine base is bound in a region of the protein which is inaccessible to collisions with these reagents. Since we have previously shown that the fluorescence of ε-ATP is quenched by water, the long lifetime of ε-ATP bound to G-actin (36 nsec, vs 27 nsec for ε-ATP in water) indicates that the bound nucleotide base is inaccessible to collisional quenching by water molecules.

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    The initial value of the identified single lifetime decreased from 24.52 ± 0.01 ns (χ2 = 2.41; n = 3) to 1.41 ± 0.03 ns (χ2 = 2.63; n = 3) in the presence of 0.3 M acrylamide. Previous results also showed a single lifetime component (27 ns) for the free ɛ-ATP that could be effectively quenched by collisional processes (62). The analysis of the results showed that the dynamic quenching constant is 54.05 ± 1.02 M−1 (n = 3) that is nearly identical to the value (53.6 M−1) that was calculated in the case of the steady-state measurements.

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