Fig 3. Rigidity of the AN1-ZnF Domain.

A) ¹⁵N-HMQC spectrum of histidine residue side-chain correlations showing the different tautomeric state patterns of zinc-coordinating His42 (blue) and His48 (red). Note the appearance of the His48 Hδ1 resonance peak that is protected from rapid exchanging with H₂O by a side-chain-to-main-chain hydrogen bond. B) Atomic model of the zinc-coordinating histidine tautomeric states. The black dotted line indicates the side-chain-to-main-chain hydrogen bond involving His42 and His48. C) 2D-NOESY spectra of the Cuz1 ZnF showing chemical exchange cross-peaks between Hδ1/Hδ2 and Hε1/Hε2 spins of Phe38 due to the slow ring-flipping rate, marked by the red dotted lines. Note that the peaks are more broadened at 45°C than at 25°C due to faster chemical exchange, consistent with increasing ring-flipping rates.