Kinetic Stability of the
Streptavidin–Biotin
Interaction Enhanced in the Gas Phase
- Publication date
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Abstract
Results of the first detailed study of the structure
and kinetic
stability of the model high-affinity protein–ligand interaction
between biotin (B) and the homotetrameric protein complex streptavidin
(S<sub>4</sub>) in the gas phase are described. Collision cross sections
(Ω) measured for protonated gaseous ions of free and ligand-bound
truncated (residues 13–139) wild-type (WT) streptavidin, i.e.,
S<sub>4</sub><sup><i>n</i>+</sup> and (S<sub>4</sub>+4B)<sup><i>n</i>+</sup> at charge states <i>n</i> = 12–16,
were found to be independent of charge state and in agreement (within
10%) with values estimated for crystal structures reported for S<sub>4</sub> and (S<sub>4</sub>+4B). These results suggest that significant
structural changes do not occur upon transfer of the complexes from
solution to the gas phase by electrospray ionization. Temperature-dependent
rate constants were measured for the loss of B from the protonated
(S<sub>4</sub>+4B)<sup><i>n</i>+</sup> ions. Over the temperature
range investigated, the kinetic stability increases with decreasing
charge state, from <i>n</i> = 16 to 13, but is indistinguishable
for <i>n</i> = 12 and 13. A comparison of the activation
energies (<i>E</i><sub>a</sub>) measured for the loss of
B from the (S<sub>4</sub>+4B)<sup>13+</sup> ions composed of WT streptavidin
and five binding site mutants (Trp79Phe, Trp108Phe, Trp120Phe, Ser27Ala,
and Tyr43Ala) suggests that at least some of the specific intermolecular
interactions are preserved in the gas phase. The results of molecular
dynamics simulations performed on WT (S<sub>4</sub>+4B)<sup>12+</sup> ions with different charge configurations support this conclusion.
The most significant finding of this study is that the gaseous WT
(S<sub>4</sub>+4B)<sup><i>n</i>+</sup> ions at <i>n</i> = 12–14, owing to a much larger <i>E</i><sub>a</sub> (by as much as 13 kcal mol<sup>–1</sup>) for the loss of
B, are dramatically more stable kinetically at 25 °C than the
(S<sub>4</sub>+4B) complex in aqueous neutral solution. The differences
in <i>E</i><sub>a</sub> values measured for the gaseous
(S<sub>4</sub>+4B)<sup><i>n</i>+</sup> ions and solvated
(S<sub>4</sub>+4B) complex can be largely accounted for by a late
dissociative transition state and the rehydration of B and the protein
binding cavity in solution