High-resolution nuclear magnetic resonance studies of the lac repressor. 1. Assignments of tyrosine resonances in the N-terminal headpiece

Abstract

The DNA binding site of the lac repressor protein (of Escherichia coli) has been implicated to lie within the N-terminal 51 amino acid fragment termed headpiece (HP-51 or LR-51). High resolution NMR suggests that isolated HP-51 retains most of the secondary and tertiary structure which it has in the whole repressor. Of the 8 tyrosines of the repressor, 4 are in HP-51. PMR spectra (360 MHz) over the aromatic region of native HP-51 show that the 4 tyrosines are nonequivalent with an unusual distribution of chemical shifts. Denaturation leads to loss of these chemical shift differences. Homonuclear decoupling and a 2-dimensional autocorrelated spectrum allow unequivocal pairing of resonances from Tyr A at 6.99 and 6.79 ppm, Tyr B at 6.98 and 6.39 ppm, Tyr C at 6.70 and 6.54 ppm, and Tyr D at 6.39 and 6.33 ppm. The 2,6 protons are low field of the 3,5 protons for each Tyr residue. Selective chemical modification with nitration reagents allows assignments of Tyr A to Tyr-47, Tyr B to Tyr-7, Tyr C to Tyr-12, and Tyr D to Tyr-17 in HP-51. All 4 tyrosines are essential for maintaining the structure of the isolated headpiece, and Tyr-7, -12 and -17 appear to be stacked.