Two-Dimensional¹H,¹⁵N NMR investigation of uniformly¹⁵N-labeledLacRepressor Headpiece

Abstract

15N uniformly labeled lac repressor and lac repressor headpiece were prepared. 15N NMR spectra of lac repressor were shown resolution inadequate for detailed study while the data showed that the 15N 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 1H and nitrogen 15NH backbone resonances of this headpiece in the free state. Assignments of the 15N 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 15NH(i)-N1H(i + 1) and 15NH(i)-N1H(i - 1) connectivities. Values of the 3JNH alpha splitting for 39 of the 51 residues of the headpiece were extracted from HMQC and HMQC-J. The observed 15NH(i)-C beta H cross peaks and the 3JNH 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 3JNH 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 3JNH alpha values, the geometry of the turn of the helix-turn-helix motif is discussed.

Backbone and Side Chain Dynamics of lac Repressor Headpiece (1−56) and Its Complex with DNA

“…No NH cross-peak was found for Met-1; residues 3 and 49 are prolines. About 20% of the assignments were different from those reported by Lancelot et al (1992) for lac HP51. Three 15 N resonances of DNA-bound lac HP56 could not be assigned due to extensive line-broadening (Table 1): these are the crosspeaks of the backbone N R H's of The 1 H resonances of lac headpiece in free and DNAbound states have been assigned earlier (Zuiderweg et al, 1983(Zuiderweg et al, , 1985Lamerichs, 1989Lamerichs, , 1990.…”

Lac repressor–operator interaction: N-terminal peptide backbone1H and15N chemical shifts upon complex formation with DNA

“…Homology with the purine repressor also suggests that a hinge helix used in DNA binding could exist from residues 50-58 (Schumacher et al, 1994). As also observed by Lancelot et al (1992), 'H/"N heteronuclear spectroscopy provides a means of substantially extending solution NMR experiments applied to lac N-terminal 'headpieces' beyond what was possible only with 'H homonuclear experiments (Kaptein et al, 1990) or "N homonuclear experiments (Bogusky et al, 1990). Using two-dimensional HMQC (Bax et al, 1983;Bax and Subramanian, 1986), 'H/"N correlations between amide protons and backbone nitrogens and from the protons on "N containing side chains are obtained.…”

“…Despite the salt concentration of 700 mM KC1 (Lancelot et al, 1992) which is over three times that used for our work, 200 mM KC1, the pattern of the correlations for the proteolytic 51 residue fragment and the cloned HP56WT peptide are sufficiently similar to enable the assignment of 44 out of 54 amide 'H-"N correlations (this count excludes Pro 3 and Pro 49) by inspection. The resonances for Lys2, Leu 6, and Tyr 17 do not appear in the HMQC spectrum of HP56WT in Fig.…”

“…Most of the assigned amino acids with shifted amide correlations upon complexation are located in helix 11, helix, 111, and the intervening turn of the N-terminal DNA binding fragment. Assignment of the correlation spectrum from HP64tyr3 was also achieved by comparison with the assignment of Lancelot et al (1992) for the proteolytic 51 amino acid headpiece (data not shown). Addition of the 11 base-pair lac operator half-site to HP64tyr3 results in similar shifts as observed for HP56WT.…”