Sequence-Specific and Stereospecific Assignment of Methyl Groups Using Paramagnetic Lanthanides
2007-11-07T00:00:00Z (GMT) by
Pseudocontact 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 εCH<sub>3</sub> 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 C<i><sub>z</sub></i>-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 <sup>13</sup>C-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 <i>Escherichia coli</i> DNA polymerase III.