Theoretical Studies of Salt-Bridge Formation by Amino Acid Side Chains in Low and Medium Polarity Environments Peter I. Nagy* ,†,‡ and Paul W. Erhardt †,‡ Center for Drug Design and DeVelopment, The UniVersity of Toledo, Toledo, Ohio 43606-3390, United States, and Department of Medicinal and Biological Chemistry, The UniVersity of Toledo, Ohio 43606, United States ReceiVed: April 13, 2010; ReVised Manuscript ReceiVed: October 13, 2010 Salt-bridge formation between Asp/Glu ··· Lys and Asp/Glu ··· Arg side chains has been studied by model systems including formic and acetic acids as proton donors and methylamine, guanidine, and methylguanidine as proton acceptors. Calculations have been performed up to the CCSD(T) CBS //MP2/aug-cc-pvtz level with formic acid proton donors. Complexes formed with acetic acid were studied at the CCSD(T)/aug-cc-pvdz// MP2/aug-cc-pvdz level. Protein environments of low and moderate polarity were mimicked by a continuum solvent with dielectric constants (ε) set to 5 and 15, respectively. Free energy differences, ∆G tot , were calculated for the neutral, hydrogen-bonded form and for the tautomeric ion pair. These values predict that a salt bridge is not favored for the Asp/Glu ··· Lys pair, even in an environment with ε as large as 15. In contrast, the Asp/Glu ··· Arg salt bridge is feasible even in an environment with ε ) 5. Charge transfers for the complexes were calculated on the basis of CHELPG and AIM charges. Introduction The protonation state of ionizable amino-acid side chains is an intricate problem in low polarity environments; namely, when imbedded within a protein. If the side chains of Asp, Glu, Arg, or Lys reside in the surrounding aqueous solution, they are expected to be mostly ionized. Determination of their proton- ation states becomes difficult, however, when the side chains are buried within the protein, where the effective dielectric constant (ε) of the environment is fairly low (e.g., ε ) 5-15). Calculation of absolute or relative pK a ’s of small molecules has been the subject of many theoretical papers in the past decades. 1-9 A more complicated problem is the determination of the protonation state of ionizable amino acid side chains in the depth of a protein. Interactions of these side chains with each other and considering the polarization effect of the environment has been studied using different theoretical methods based mainly on continuum solvent approaches. 10-17 Recent experimental studies, including 1 H, 13 C, 15 N NMR methods; UV/ vis spectrophotometry; and neutron crystallography, have provided data for comparison with the theoretical results. 18-21 The general conclusions from the theoretical studies emphasize that geometric data and conformational issues as well as molecular mechanic force fields and parametrizations 22-27 have sensitive effect on the obtained results. In some cases, the Asp or Glu side chain faces an Arg or Lys side chain in a protein such that two kinds of strong interactions are possible. If the residues adopt their neutral forms, then a hydrogen bond becomes favorable at a proper separation of the polar centers. An alternative interaction is possible when this complex adopts a so-called “salt-bridge” arrangement. A salt bridge corresponds to a hydrogen-bonded form, as well, but the partners are formally ionic (Scheme 1). This situation becomes important during molecular-mechanics-based modeling in which the formal charges for the residues are preset and do not change throughout the simulation. The formal atomic charges are part of the applied force field and must be determined in harmony with other force-field parameters. In a force field by Duan et al., 28 atomic charges * Corresponding author. E-mail: pnagy@utnet.utoledo.edu. † Center for Drug Design and Development. ‡ Department of Medicinal and Biological Chemistry. SCHEME 1: FA ··· MeNH 2 (1a), FA - ··· MeNH 3 + (1b), FA ··· Gua (2aa), FA ··· Gua (2ab), FA - ··· GuaH + (2b), AA ··· MeNH 2 (3a), AA - ··· MeNH 3 + (3b), AA ··· MeGua (4a), AA - ··· MeGuaH + (4b), Propanoic acid (5a), and Propanoate (5b) J. Phys. Chem. B 2010, 114, 16436–16442 16436 10.1021/jp103313s  2010 American Chemical Society Published on Web 11/19/2010