Sequence-specific and stereospecific assignment of methyl groups using paramagnetic lanthanides.
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 epsilonCH3 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 epsilon and the subunit theta of the Escherichia coli DNA polymerase III.
John
Michael
M
Research School of Chemistry, Australian National University, Canberra, ACT 0200, Australia.
Schmitz
Christophe
C
Park
Ah Young
AY
Dixon
Nicholas E
NE
Huber
Thomas
T
Otting
Gottfried
G
D001665:Binding Sites; D002247:Carbon Isotopes; D004258:DNA Polymerase III / Q000737:chemistry*; D004259:DNA-Directed DNA Polymerase / Q000737:chemistry*; D004926:Escherichia coli / Q000737:chemistry; D029968:Escherichia coli Proteins / Q000737:chemistry*; D028581:Lanthanoid Series Elements / Q000737:chemistry*; D009682:Magnetic Resonance Spectroscopy / Q000379:methods / Q000592:standards; D008280:Magnetics; D012015:Reference Standards; D012680:Sensitivity and Specificity; D013237:Stereoisomerism; D013379:Substrate Specificity
D002247:Carbon Isotopes; D029968:Escherichia coli Proteins; D028581:Lanthanoid Series Elements; C505238:holE protein, E coli; D004258:DNA Polymerase III; D004259:DNA-Directed DNA Polymerase