Electrostatic calculations for assignment of infrared difference bands to carboxyl groups getting protonated during protein reactions.

Fourier transform infrared (FTIR) difference spectroscopy is predestined to monitor the protonation of carboxyl groups during protein reactions, making glutamic and aspartic amino acids unique to follow proton pathways. The absorption of the corresponding vibrations are clearly distinguishable from the absorption of other amino acids. However, the assignment to specific groups within the protein needs additional information, e.g., from induced spectral changes due to isotopic labeling or mutation. Here, the capability of electrostatic calculations to assign IR difference bands to specific carboxyl groups getting protonated is demonstrated by the ion pump mechanism of the sarcoplasmic reticulum Ca(2+)-ATPase. Active Ca(2+) transport is coupled to the hydrolysis of ATP. Two Ca(2+) ions are transported per ATP hydrolysed and two or three H(+) ions are countertransported. FTIR difference spectra show that during the Ca(2+) release step, carboxyl groups become protonated. Multiconformation continuum electrostatic calculations (MCCE) have been carried out to determine the equilibrium distribution of residue ionization and side chain conformation in dependence of pH. Available structural X-ray data from the calcium-bound and the calcium-free state allows us to simulate the transition between the two states monitored in the IR difference spectra. Exemplarily for Asp 800, ligand of both calcium ions, it is shown that MCCE calculations can identify this specific Asp to contribute to the IR bands and therefore to take part in the proton countertransport of the Ca(2+)-ATPase. In addition, an energy analysis can be performed to understand what interactions shift the pK(a).