Flexibility and dynamics of NhaA Na+/H+-antiporter of Escherichia coli studied by Fourier transform infrared spectroscopy.

Abstract NhaA (41,355 Da) is a Na + /H + antiporter of Escherichia coli which plays a central role in regulation of intracellular pH, cellular Na + content, and cell volume [E. Padan, S. Schuldiner, J. Exp. Biol. 196 (1994) 443]. Its activity is strongly regulated by pH and increases over 3 orders of magnitude between pH 7 and 8 [A. Rothman, Y. Gerchman, E. Padan, S. Schuldiner, Biochemistry 36 (1997) 14572]. Protein dynamics and flexibility in the activated and inactivated state, respectively, was analysed by probing accessibility in 1 H/ 2 H exchange experiments for the wild type and the mutant G338S which is constitutively active independent of pH [A. Rimon, Y. Gerchman, Z. Kariv, E. Padan, J. Biol. Chem. 273 (1998) 26470]. This was studied by ATR–FTIR difference spectroscopy using a home built microvolume ( On the basis of 1 H– 2 H exchange of NhaA followed in the amide I and amide II region of the IR spectrum, it is concluded that the accessible fraction of the polypeptide chain of NhaA increases by more than 10% in the active state. For the mutant, no changes in accessibility were observed for different pH values. The increase of Na + concentration increases the extent of exchange. The stability of the wild type protein in the active and inactive form was analysed by measuring the temperature profiles of the IR spectra. A decrease of the structural stability of the protein with activation was observed. Together with the results from 1 H/ 2 H exchange, the inactive state represents a more compact form whereas activation induces a more open conformation of the protein.