Methyl group assignment using pseudocontact shifts with PARAssign

Mathilde Lescanne,Claudio Luchinat,Marcellus Ubbink,Anneloes Blok,Simon P Skinner,Marco Fragai,Linda Cerofolini,Monika Timmer

doi:10.1007/s10858-017-0136-3

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.

Highlights

Liquid state NMR is widely used to study protein structure, dynamics and interactions with atomic detail
Individual domains or subunits are first assigned, followed by a transfer of assignments to the spectrum of the larger multidomain protein or multisubunit protein complex (Mas et al 2013; Gelis et al 2007; Velyvis et al 2009). Another approach is to use multidimensional NOESY spectra in combination with a crystal structure (Venditti et al 2011). This approach has been implemented in the MAP-XSII software (Xu and Matthews 2013) that automatically assigns methyl groups based on a known structure combined with NOEs measured on 4D NOESY experiments
Assignment based on pseudocontact shifts (PCS) requires accurate PCS that are not affected by averaging effects due to tag mobility

Summary

Introduction

Liquid state NMR is widely used to study protein structure, dynamics and interactions with atomic detail. Individual domains or subunits are first assigned, followed by a transfer of assignments to the spectrum of the larger multidomain protein or multisubunit protein complex (Mas et al 2013; Gelis et al 2007; Velyvis et al 2009). Another approach is to use multidimensional NOESY spectra in combination with a crystal structure (Venditti et al 2011). The program FLAMEnGO uses a combination of 3D NOESY, methine–methyl TOCSY and paramagnetic relaxation enhancements (PREs) and a protein structure to assign methyl groups (Chao et al 2014)

Methods

Results

Conclusion