Assignment of N-15, C-13(alpha), C-13(beta), and HN resonances in an N-15, C-13, H-2 labeled 64 kDa trp repressor-operator complex using triple-resonance NMR spectroscopy and H-2-decoupling

The near complete (>90%) NMR assignment of N-15, C-13(alpha), C-13(beta) and HN chemical shifts is presented for a 64 kDa trp repressor-operator complex consisting of two tandem dimers of N-15,C-13,>90% H-2 labeled trp repressor, unlabeled 22-base-pair DNA, and unlabeled corepressor, 5-methyltryptophan. The DNA sequence employed contains three copies of the palindromic sequence S'-CTAG-3', allowing two dimers of trp repressor to bind to each duplex operator DNA. Chemical shift: data establish thar, each subunit within a given dimer in the complex is in a chemically distinct environment, and the pattern of chemical shift differences between subunits provides information regarding interdimer contacts. Because of the large size of the complex, a number of modifications were made to existing enhanced sensitivity triple-resonance correlation experiments which link C-13(beta), N-15, and HN chemical shifts; the pulse sequences which include these changes are presented. The experiments make use of constant-time chemical shift evolution of the carbon magnetization, resulting in significant improvements in spectral resolution compared to non-constant-time versions of the pulse schemes. An analysis of the utility of the enhanced sensitivity method for recording spectra of high molecular weight deuterated proteins indicates that this approach produces reasonable sensitivity gains for the 64 kDa trp repressor-operator complex studied here.