Methyl group assignment using pseudocontact shifts with PARAssign

Abstract

A new version of the program PARAssign has been evaluated for assignment of NMR resonances of the 76 methyl groups in leucines, isoleucines and valines in a 25 kDa protein, using only the structure of the protein and pseudocontact shifts (PCS) generated with a lanthanoid tag at up to three attachment sites. The number of reliable assignments depends strongly on two factors. The principle axes of the magnetic susceptibility tensors of the paramagnetic centers should not be parallel so as to avoid correlated PCS. Second, the fraction of resonances in the spectrum of a paramagnetic sample that can be paired with the diamagnetic counterparts is critical for the assignment. With the data from two tag positions a reliable assignment could be obtained for 60% of the methyl groups and for many of the remaining resonances the number of possible assignments is limited to two or three. With a single tag, reliable assignments can be obtained for methyl groups with large PCS near the tag. It is concluded that assignment of methyl group resonances by paramagnetic tagging can be particularly useful in combination with some additional data, such as from mutagenesis or NOE-based experiments. Approaches to yield the best assignment results with PCS generating tags are discussed.Electronic supplementary materialThe online version of this article (doi:10.1007/s10858-017-0136-3) contains supplementary material, which is available to authorized users.

Methyl group reorientation under ligand binding probed by pseudocontact shifts

“…Ntd-HSP90 was tagged at three sites using the Caged Lanthanoid NMR probe #5 (CLaNP-5), containing either Lu 3+ as a diamagnetic control or Yb 3+ as a paramagnetic center. The tagging sites, double mutants 50C/54C, 101C/105C and 149C/187C, have been described before (Lescanne et al 2017 ). Assignments are shown in Fig.…”

“…S1. Methyl 13 C- 1 H HSQC spectra of WT and CLaNP-5(Lu 3+ ) tagged mutants are very similar except for the resonance of a few methyl groups very close to the tags (Lescanne et al 2017 ), indicating that the tags do not have large effects on the structure of the protein. A first comparison of the methyl group spectra of the paramagnetic and diamagnetic samples illustrates the increased dispersion of the resonances for the paramagnetic samples (Fig.…”

“…Three double cysteine mutants of the ntd-HSP90 were designed on the surface of the protein, S50C/D54C, A101C/N105C and T149C/I187C (Lescanne et al 2017 ). Ntd-HSP90 does not have any native cysteines.…”

“…Ntd-HSP90 does not have any native cysteines. The protein was produced labelled with Leu-δ1-δ2/Val-γ1-γ2-[ 13 CH 3 ] and purified and tagged with CLaNP5 according to a published protocol (Lescanne et al 2017 ). CLaNP-5 was synthesized as described before (Keizers et al 2007 , 2008 ).…”

“…Methyl groups assignments have been performed before with traditional through-backbone NMR techniques and confirmed by PARAssign (Skinner et al 2013 ; Lescanne et al 2017 ). PARAssign provided the stereo-specific assignment with high reliability for 14 Leu/Val methyl groups (Lescanne et al 2017 ), based on PCS generated by CLaNP-5 attached at two distinct positions, S50C/D54C and A101C/N105C.…”