Towards unambiguous assignment of methyl-containing residues by double and triple sensitivity-enhanced HCCmHm-TOCSY experiments.
Chemical shift assignment of methyl-containing residues is essential in protein NMR spectroscopy, as these residues are abundant in protein interiors and provide the vast majority of long-range NOE connectivities for structure determination. These residues also constitute an integral part of hydrophobic cavities, the surroundings for many enzymatic reactions. Here we present a powerful strategy for the assignment of methyl-containing residues in a uniformly 13C/15N double labeled protein sample. The approach is based on novel four-dimensional HCCmHm-TOCSY experiments, two of them utilizing gradient selection and sensitivity enhancement in all three indirectly detected dimensions. Regardless of the number of dimensions, the proposed experiments can be executed using only one transient per FID, providing outstanding resolution and sensitivity. A complete assignment of the 51 methyl-containing residues in the 16 kDa Mus musculus coactosin was accomplished using a four-dimensional HCCmHm-TOCSY spectrum recorded in 16 hours.
Würtz
Peter
P
Program in Structural Biology and Biophysics, Institute of Biotechnology/NMR Laboratory, University of Helsinki, FI-00014, Helsinki, Finland.
Hellman
Maarit
M
Tossavainen
Helena
H
Permi
Perttu
P
D000595:Amino Acid Sequence; D000818:Animals; D051379:Mice; D008840:Microfilament Proteins / Q000737:chemistry*; D008970:Molecular Weight; D019906:Nuclear Magnetic Resonance, Biomolecular; D012680:Sensitivity and Specificity; D013997:Time Factors
C485551:Cotl1 protein, mouse; D008840:Microfilament Proteins