2-DIMENSIONAL H-1, N-15 NMR INVESTIGATION OF UNIFORMLY N-15-LABELED LAC REPRESSOR HEADPIECE

N-15 uniformly labeled lac repressor and lac repressor headpiece were prepared. N-15 NMR spectra of lac repressor were shown resolution inadequate for detailed study while the data showed that the N-15 labeled N-terminal part of the protein is quite suitable for this type of study allowing future investigation of the specific interaction of the lac repressor headpiece with the lac operator. We report here the total assignment of proton H-1 and nitrogen (NH)-N-15 backbone resonances of this headpiece in the free state. Assignments of the N-15 resonances of the protein were obtained in a sequential manner using heteronuclear multiple quantum coherence (HMQC), relayed HMQC nuclear Overhauser and relayed HMQC-HOHAHA spectroscopy. More than 80 per cent of residues were assigned by their (NH(i)-NH(i+1)-N-15-H-1 and (NH(i)-NH(i-1)-N-15-H-1 connectivities. Values of the 3J(NH-alpha) splitting for 39 of the 51 residues of the headpiece were extracted from HMQC and HMQC-J. The observed (NH(i)-C(beta)H)-N-15 cross peaks and the 3J(NH-alpha) coupling constants values are in agreement with the three alpha-helices previously described [Zuiderweg, E.R.P., Scheek, R.M., Boelens, R., van Gunsteren, W.F. and Kaptein, R., Biochimie 67, 707 (1985)]. The 3J(NH-alpha) coupling constants can be now used for a more confident determination of the lac repressor headpiece. From these values it is shown that the geometry of the ends of the second and third alpha-helices exhibit deviation from the canonical alpha-helix structure. On the basis of NOEs and 3J(NH-alpha) values, the geometry of the turn of the helix-turn-helix motif is discussed.