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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.