ja0744753_si_001.pdf (1.86 MB)
Sequence-Specific and Stereospecific Assignment of Methyl Groups Using Paramagnetic Lanthanides
journal contribution
posted on 2007-11-07, 00:00 authored by Michael John, Christophe Schmitz, Ah Young Park, Nicholas E. Dixon, Thomas Huber, Gottfried OttingPseudocontact shifts (PCSs) induced by a site-specifically bound paramagnetic lanthanide ion
are shown to provide fast access to sequence-specific resonance assignments of methyl groups in proteins
of known three-dimensional structure. Stereospecific assignments of Val and Leu methyls are obtained as
well as resonance assignments of all other methyls, including Met εCH3 groups. No prior assignments of
the diamagnetic protein are required nor are experiments that transfer magnetization between the methyl
groups and the protein backbone. Methyl Cz-exchange experiments were designed to provide convenient
access to PCS measurements in situations where a paramagnetic lanthanide is in exchange with a
diamagnetic lanthanide. In the absence of exchange, simultaneous 13C-HSQC assignments and PCS
measurements are delivered by the newly developed program Possum. The approaches are demonstrated
with the complex between the N-terminal domain of the subunit ε and the subunit θ of the Escherichia coli
DNA polymerase III.