Solid State NMR Structure-Function Analysis of the Stress-Response Peptide TisB

Abstract

The 29-residue peptide TisB is expressed in E.coli in response to stress and has been suggested to break down the transmembrane potential by equilibrating the proton gradient [1,2]. We characterized its membrane bound structure using solid-state NMR and oriented CD (OCD). For the NMR analysis we individually substituted several hydrophobic amino acids in TisB with CF3-L-Bpg, and/or with a 15N-label. CD analysis showed that all analogues remain α-helical, similar to the wild type peptide. 19F-NMR showed that TisB assumes a transmembrane orientation, with its helix aligned parallel to the membrane normal, as confirmed by 15N-NMR and OCD. For a functional characterization we used a vesicle based fluorescence assay to test the breakdown of the pH gradient. The rate of pH equilibration as a function of peptide concentration revealed a Hill coefficient of 2, which is suggestive of dimer formation. This gave rise to the idea that an antiparallel dimer could be formed and stabilized in the membrane by four intermolecular salt bridges and one central H-bond at Gln19. To test this hypothesis, we synthesized several TisB mutants in which the charged residues engaged in the postulated salt bridges were mutually exchanged, and where Gln19 was modified. The functional activities of these peptide mutants confirmed that TisB assembles via an electrostatic “charge zipper” [3] in the membrane and is thereby able to transport H+ or OH- ions.[1] Steinbrecher et al. (2012) Biophys. J., 103: 1460-1469.[2] Berditsch et al. (2012) Front. Immun. 3:222.[3] Walther et al. (2013) Cell, 152: 316-326.