Direct NMR detection of bifurcated hydrogen bonding in the ?-helix N-caps of ankyrin repeat proteins.

ABSTRACT: In biomolecules, bifurcated H-bonds typically involve the interaction of two donor protons with the two lone pairs of oxygen. Here, we present direct NMR evidence for a bifurcated H-bonding arrangement involving nitrogen as the acceptor atom. Specifically, the H-bond network comprises the N?1 atom of histidine and both the backbone N-H and side-chain O?-H of threonine within the conserved TXXH motif of ankyrin repeat (AR) proteins. Identification of the H-bonding partners is achieved via solution NMR H-bond scalar coupling (HBC) and H/D isotope shift experiments. Quantitative determination of (2h)J(NN) HBCs supports that Thr N-H···N?1 His H-bonds within internal repeats are stronger (?4 Hz) than in the solvent exposed C-terminal AR (?2 Hz). In agreement, pKa values for the buried histidines bridging internal ARs are several units lower than those of the C-terminus. Quantum chemical calculations show that the relevant (2h)J and (1h)J couplings are dominated by the Fermi contact interaction. Finally, a Thr-to-Val replacement, which eliminates the Thr O?-H···N?1 His H-bond and decreases protein stability, results in a 25% increase in (2h)J(NN), attributed to optimization of the Val N-H···N?1 His H-bond. Overall, the results provide new insights into the H-bonding properties of histidine, a refined structural rationalization for the folding cooperativity of AR proteins, and a challenging benchmark for the calculation of HBCs.

SUBMITTER: Preimesberger MR 

PROVIDER: S-EPMC4311973 | biostudies-literature | 2015 Jan

REPOSITORIES: biostudies-literature