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Direct NMR Detection of Bifurcated Hydrogen Bonding in the α-Helix N-Caps of Ankyrin Repeat Proteins

Preimesberger, Matthew R., Majumdar, Ananya, Aksel, Tural, Sforza, Kevin, Lectka, Thomas, Barrick, Doug, Lecomte, Juliette T. J.
Journal of the American Chemical Society 2015 v.137 no.3 pp. 1008-1011
histidine, hydrogen bonding, isotopes, nitrogen, nuclear magnetic resonance spectroscopy, oxygen, proteins, protons, quantitative analysis, solvents, threonine
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 Nδ1 atom of histidine and both the backbone N–H and side-chain Oγ-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 ²ʰJNN HBCs supports that Thr N–H···Nδ1 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 ²ʰJ and ¹ʰJ couplings are dominated by the Fermi contact interaction. Finally, a Thr-to-Val replacement, which eliminates the Thr Oγ-H···Nδ1 His H-bond and decreases protein stability, results in a 25% increase in ²ʰJNN, attributed to optimization of the Val N–H···Nδ1 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.