Subtle Dynamics of holo Glutamine Binding Protein Revealed with a Rigid Paramagnetic Probe

Bacterial periplasmic binding proteins (PBPs) are involved in the translocation of small molecules in the periplasm. To unload, the two domains of a PBP open up, allowing the ligand to exit. However, it is not clear whether there are dynamics near the binding site which can facilitate the rapid dissociation of a ligand. To visualize such dynamics, we utilized paramagnetic relaxation enhancement (PRE) NMR and introduced a rigid paramagnetic probe to a PBP, glutamine-binding protein (QBP) with its cognate ligand bound. A paramagnetic Cu(II) ion is sandwiched between an engineered di-histidine motif at a helix and an NTA capping molecule. The afforded paramagnetic probe is so rigid that PRE values calculated from a single structure of holo QBP largely agree with the observed values. The remaining PRE discrepancies, however, manifest dynamics of a loop in the opposite domain from the paramagnetic probe. This loop packs against the glutamine ligand in the holo QBP and undergoes fluctuations upon ligand dissociation, as assessed by steered molecular dynamics simulations. As such, the loop dynamics, occurring for a small population in nanosecond to microsecond time scale, may be related to the ligand dissociation process. The rigid paramagnetic probe described herein can be grafted to other protein systems for structure and dynamics studies.