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

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 1H/2H 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 (1H–2H 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 1H/2H exchange, the inactive state represents a more compact form whereas activation induces a more open conformation of the protein

Infrared spectroscopic study of the structural and functional properties of the Na+/H+ antiporter MjNhaP1 from Methanococcus jannaschii

AbstractIn this study, structural, functional, and mechanistic properties of the Na+/H+ antiporter MjNhaP1 from Methanococcus jannaschii were analyzed by infrared spectroscopic techniques. Na+/H+ antiporters are generally responsible for the regulation of cytoplasmic pH and Na+ concentration. MjNhaP1 is active in the pH range between pH 6 and pH 6.5; below and above it is inactive.The secondary structure analysis on the basis of ATR-IR spectra provides the first insights into the structural changes between inactive (pH 8) and active (pH 6) state of MjNhaP1. It results in decreased ordered structural elements with increasing the pH-value i.e. with inactivation of the protein. Analysis of temperature-dependent FTIR spectra indicates that MjNhaP1 in the active state exhibits a much higher unfolding temperature in the spectral region assigned to α-helical segments. In contrast, the temperature-induced structural changes for β-sheet structure are similar for inactive and active state. Consequently, this structure element is not the part of the activation region of the protein. The surface accessibility of the protein was analyzed by following the extent of H/D exchange. Due to higher content of unordered structural elements a higher accessibility for amide protons is observed for the inactive as compared to the active state of MjNhaP1. Altogether, the results present the active state of MjNhaP1 as the state with ordered structural elements which exhibit high thermal stability and increased hydrophobicity