Conformation and Metal Cation Binding of Zwitterionic Alanine Tripeptide in Saline Solutions by Infrared Vibrational Spectroscopy and Molecular Dynamics Simulations

In this work, linear infrared (IR) spectroscopy and molecular dynamics (MD) simulations were used to examine the interaction of different metal cations (Na+, Ca2+, Mg2+, and Zn2+) with backbone (amide C=O) and C-terminal carboxylate (COO-) groups in zwitterionic alanine tripeptide (Ala3) in aqueous solutions with varying saline concentrations. Circular dichroism spectra and MD results suggest that Ala3 is predominantly in polyproline-II (PPII) conformation, whose amide-I and asymmetric carboxylate stretching IR vibration signatures are also supported by quantum-chemistry calculations. The zwitterionic form of Ala3 separates the two amide-I modes in frequency, which are weakly coupled modes, as revealed by two-dimensional IR measurement, and can be used to probe backbone-cation interactions at different scenarios (near charged or neutral chemical groups respectively). Cation concentration-dependent IR frequency red shifts in the amide-I mode are seen for both amide-I modes, whereas blue shifts are also seen in the amide-I mode far from the NH3+ group. The observed spectral changes are discussed from the perspective of the salting-in and salting-out abilities of the cations. In addition, all the metal cations studied here (except Zn2+) can specifically coordinate to the COO- group in bidentate and pseudo-bridging forms simultaneously. For Zn2+, only the pseudo-bridging form exists. Our results shed light on the macroscopic protein salting-in and salting-out phenomena from the perspective of key chemical bonds in peptides.