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The energetics of protonation of pyridine and a series of its derivatives, as well as the energetics of formation of hydrogen-bonded N...H...N homocomplexed cations in systems involving substituted pyridines and conjugate cationic acids were investigated by means of restricted Hartree-Fock (RHF) and Møller-Plesset (MP2) ab initio calculations. The Gaussian functional basis set 6-31G* was employed to calculate energy and Gibbs free energy of protonation and cationic homoconjugation in the gas phase. The proton potential of a homocomplexed pyridine cation exhibits a double minimum with a 5.7 kcal/mol energy barrier, which could be reduced to 2.7 kcal/mol by accounting for a thermodynamic correction factor. The calculated protonation energies, ∆E prot , and Gibbs free energies, ∆G prot , have been found to correlate well with the acid dissociation constants (expressed as pK a values) in acetonitrile. On the contrary, the calculated energies, ∆E BHB + , and Gibbs free energies, ∆G BHB + , of formation of the homocomplexes do not correlate with the cationic homoconjugation constant values (expressed as log 10 K BHB + ) determined in acetonitrile.

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Dr fimbriae are homopolymeric adhesive organelles of uropathogenic Escherichia coli composed of DraE subunits, responsible for the attachment to host cells. These structures are characterized by enormously high stability resulting from the structural properties of an Ig-like fold of DraE. One feature of DraE and other fimbrial subunits that makes them peculiar among Ig-like domain-containing proteins is a conserved disulfide bond that joins their A and B strands. Here, we investigated how this disulfide bond affects the stability and folding/unfolding pathway of DraE. We found that the disulfide bond stabilizes self-complemented DraE (DraE-sc) by ∼50 kJ mol−1 in an exclusively thermodynamic manner, i.e. by lowering the free energy of the native state and with almost no effect on the free energy of the transition state. This finding was confirmed by experimentally determined folding and unfolding rate constants of DraE-sc and a disulfide bond-lacking DraE-sc variant. Although the folding of both proteins exhibited similar kinetics, the unfolding rate constant changed upon deletion of the disulfide bond by 10 orders of magnitude, from ∼10−17 s−1 to 10−7 s−1. Molecular simulations revealed that unfolding of the disulfide bond-lacking variant is initiated by strands A or G and that disulfide bond-mediated joining of strand A to the core strand B cooperatively stabilizes the whole protein. We also show that the disulfide bond in DraE is recognized by the DraB chaperone, indicating a mechanism that precludes the incorporation of less stable, non-oxidized DraE forms into the fimbriae.

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