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-4) in the gas phase are described. Collision cross sections (Omega) measured for protonated gaseous ions of free and ligand-bound truncated (residues 13-139) wildtype (WT) streptavidin, i.e., S-4(n+) and (S-4+4B)(n+) at charge states n = 12-16, were found to be independent of charge state and in agreement (within 10%) with values estimated for crystal structures reported for S-4 and (S-4+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-4+4B)(n+) ions. Over the temperature range investigated, the kinetic stability increases with decreasing charge state, from n = 16 to 13, but is indistinguishable for n = 12 and 13. A comparison of the activation energies (E-a) measured for the loss of B from the (S-4+4B)(13+) 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-4+4B)(12+) ions with different charge configurations support this conclusion. The most significant finding of this study is that the gaseous WT (S-4+4B)(n+) ions at n = 12-14, owing to a much larger E-a (by as much as 13 kcal mol(-1)) for the loss of B, are dramatically more stable kinetically at 25 C than the (S-4+4B) complex in aqueous neutral solution. The differences in E-a values measured for the gaseous (S-4+4B)(n+) ions and solvated (S-4+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.