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(bet a) and HN chemical shifts is presented for a 64 kDa trp repressor-oper ator complex consisting of two tandem dimers of N-15,C-13,>90% H-2 lab eled trp repressor, unlabeled 22-base-pair DNA, and unlabeled corepres sor, 5-methyltryptophan. The DNA sequence employed contains three copi es of the palindromic sequence S'-CTAG-3', allowing two dimers of trp repressor to bind to each duplex operator DNA. Chemical shift: data es tablish thar, each subunit within a given dimer in the complex is in a chemically distinct environment, and the pattern of chemical shift di fferences between subunits provides information regarding interdimer c ontacts. Because of the large size of the complex, a number of modific ations were made to existing enhanced sensitivity triple-resonance cor relation experiments which link C-13(beta), N-15, and HN chemical shif ts; 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 spectr al resolution compared to non-constant-time versions of the pulse sche mes. An analysis of the utility of the enhanced sensitivity method for recording spectra of high molecular weight deuterated proteins indica tes that this approach produces reasonable sensitivity gains for the 6 4 kDa trp repressor-operator complex studied here.