title: Direct NMR detection of bifurcated hydrogen bonding in the α-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 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 (2h)J(NN) 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 (2h)J and (1h)J 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 (2h)J(NN), 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.
authors.affiliation: T. C. Jenkins Department of Biophysics and ‡Department of Chemistry, Johns Hopkins University , 3400 North Charles Street, Baltimore, Maryland 21218, United States.
authors.forename: Matthew R
authors.initials: MR
authors.lastname: Preimesberger
authors.forename: Ananya
authors.initials: A
authors.lastname: Majumdar
authors.forename: Tural
authors.initials: T
authors.lastname: Aksel
authors.forename: Kevin
authors.initials: K
authors.lastname: Sforza
authors.forename: Thomas
authors.initials: T
authors.lastname: Lectka
authors.forename: Doug
authors.initials: D
authors.lastname: Barrick
authors.forename: Juliette T J
authors.initials: JT
authors.lastname: Lecomte
mesh_terms: D017089:Ankyrin Repeat; D017487:Ankyrins / Q000737:chemistry*; D006860:Hydrogen Bonding; D008958:Models, Molecular; D019906:Nuclear Magnetic Resonance, Biomolecular; D011789:Quantum Theory
chemical_list: D017487:Ankyrins