A minor conformation of a lanthanide tag on adenylate kinase characterized by paramagnetic relaxation dispersion NMR spectroscopy

Abstract

NMR relaxation dispersion techniques provide a powerful method to study protein dynamics by characterizing lowly populated conformations that are in dynamic exchange with the major state. Paramagnetic NMR is a versatile tool for investigating the structures and dynamics of proteins. These two techniques were combined here to measure accurate and precise pseudocontact shifts of a lowly populated conformation. This method delivers valuable long-range structural restraints for higher energy conformations of macromolecules in solution. Another advantage of combining pseudocontact shifts with relaxation dispersion is the increase in the amplitude of dispersion profiles. Lowly populated states are often involved in functional processes, such as enzyme catalysis, signaling, and protein/protein interactions. The presented results also unveil a critical problem with the lanthanide tag used to generate paramagnetic relaxation dispersion effects in proteins, namely that the motions of the tag can interfere severely with the observation of protein dynamics. The two-point attached CLaNP-5 lanthanide tag was linked to adenylate kinase. From the paramagnetic relaxation dispersion only motion of the tag is observed. The data can be described accurately by a two-state model in which the protein-attached tag undergoes a 23° tilting motion on a timescale of milliseconds. The work demonstrates the large potential of paramagnetic relaxation dispersion and the challenge to improve current tags to minimize relaxation dispersion from tag movements.

Pseudocontact shifts in biomolecular NMR using paramagnetic metal tags

“…Unfortunately, attempts to enhance relaxation dispersion effects by paramagnetic lanthanide tags have so far been thwarted by difficulties distinguishing motions of the protein from those of the tag [151,152,198]. Alternatively, however, chemical exchange saturation transfer (CEST) experiments can be used to measure chemical shifts and PCSs of lowpopulated states.…”

Enantiomeric two-armed lanthanide-binding tags for complementary effects in paramagnetic NMR spectroscopy

“…Although the effective ''linkers'' between the protein-bound T1/T2 are the same as those of the CLaNP-5 and CLaNP-7 tags, the chiral elements of the T1/T2 design may help shed light on the source of the residual motions of the CLaNP-5 tag, recently reported in relaxation dispersion NMR experiments. 39 In conclusion, we have demonstrated that the enantiomeric T1/T2 tags, which can be readily synthesised in parallel, can provide long-range complementary structural information from the same tagging site, thus requiring fewer double-cysteine protein mutants for structural investigations. This reflects the fact that attachment of the tags produces a pair of diastereomeric protein-tag conjugates.…”

Protein–Protein Interactions

“…It has 29 been shown that combining relaxation dispersion with PCS data can potentially be very powerful but paramagnetic tag mobility is still a large problem, because it causes additional, undesired relaxation dispersion effects. 89,90 In conclusion, paramagnetic NMR has shown to have many applications for protein NMR and it is expected that its star will brighten in the future. PRE has been used widely, but the power of PCS and pRDC is still to be appreciated more by many in the NMR field.…”