Mobility of Histidine Side Chains Analyzed with ¹⁵N NMR Relaxation and Cross-Correlation Data: Insight into Zinc-Finger–DNA Interactions

Abstract

Due to chemical exchange, the mobility of histidine (His) side chains of proteins is typically difficult to analyze by NMR spectroscopy. Using an NMR approach that is uninfluenced by chemical exchange, we investigated internal motions of the His imidazole NH groups that directly interact with DNA phosphates in the Egr-1 zinc-finger-DNA complex. In this approach, the transverse and longitudinal cross-correlation rates for 15 N chemical shift anisotropy (CSA) and 15 N-1 H dipole-dipole relaxation interference were analyzed together with 15 N longitudinal relaxation rates and heteronuclear Overhauser effect data at two magnetic field strengths. We found that the zinc-coordinating His side chains directly interacting with DNA phosphates are strongly restricted in mobility. This makes a contrast to the arginine and lysine side chains that retain high mobility despite their interactions with DNA phosphates in the same complex. The entropic effects of side-chain mobility on molecular association are discussed.

Methodological advancements for characterising protein side chains by NMR spectroscopy

“…These exchange events have recently been probed using 13 C relaxation dispersion [44]. Alternatively, an 15 N cross correlated relaxation approach [45], originally developed for the protein backbone and unaffected by micro-millisecond motions [46], can be used to accurately report on piconanosecond motions of histidine side chains.…”