TROSY Triple-Resonance Four-Dimensional NMR Spectroscopy of a 46 ns Tumbling Protein

Two four-dimensional TROSY triple resonance based experiments are presented for backbone assignment of high molecular weight proteins and protein complexes. The experiments, 4D-HNCACO and 4D-HNCOCA, establish correlations of the form ( 13 C α ( i, i - 1), 13 C‘ ( i, i - 1), 15 N ( i ),HN ( i ) ) and ( 13 C α ( i - 1), 13 C‘ ( i - 1), 15 N ( i ),HN ( i ) ), respectively. Both sequences use an implementation of TROSY that offers improved sensitivity relative to previous sequences, critical for application to systems with correlation times on the order of 40−50 ns. The utility of the experiments is demonstrated by an application to a 46 ns tumbling complex of the 370 residue maltose binding protein and β-cyclodextrin. Approximately 95% of the expected intra- and interresidue correlations are observed in the HNCACO and HNCOCA, respectively, with average signal-to-noise values of approximately 35/1. The methodology promises to be particularly powerful for applications to high molecular weight complexes comprised of a labeled fragment and unlabeled components or proteins with segmental labeling.