Sequence‐specific NMR assignments of the trp repressor from Escherichia coli using three‐dimensional ¹⁵N/¹H heteronuclear techniques

Abstract

Sequence-specific I5N and 'H assignments for the trp holorepressor from Escherichia coli are reported. The trp repressor consists of two identical 107-residue subunits which are highly helical in the crystal state [Schevitz, R., Otwinowski, Z., Joachimiak, A., Lawson, C. L. & Sigler, P. B. (1985) Nature 31 7, 782 -7861. The high helical content and the relatively large size of the protein (Mr = 25 000) make it difficult to assign even the main-chain resonances by conventional homonuclear twodimensional NMR methods. However, we have now assigned the main-chain resonances of 94% of the residues by using three-dimensional ' 5N/'H heteronuclear experiments on a sample of protein uniformly labelled with "N. The additional resolution obtained by spreading out the signals into three dimensions proved indispensable in making these assignments. In particular, we have been able to resolve signals from residues in the N-terminal region of the A helix for the first time in solution. The observed NOE results confirm that the repressor is highly helical in solution, and contains no extended chain conformations.The trp repressor is a 25-kDa dimeric protein which controls transcription of the trpO, trpR and aroH operons in Escherirhiu coli (Platt, 1980;Klig et al., 1988). The trp repressor regulates transcription by selectively binding to a 20-base-pair operator and interfering with RNA polymerase activity. The DNA binding affinity is allosterically regulated by intracellular levels of L-tryptophan (It0 and Yanofsky, 1969).The structure of the trp repressor has been extensively studied crystallographically by Sigler and coworkers (Schevitz et al., 1985;Zhang et al., 1987;Lawson et al., 1988;Otwinowski et al., 1988). The trp repressor dimer has 107 residues/subunit ; each subunit consists of six helices separated by /j' turns. The DNA-binding domain is formed by the D and E helices which comprise the helix-turn-helix motif common to many prokaryotic and phage DNA-binding proteins (for reviews see Steitz, 1990;Freemont et al., 1991). NMR studies to date have confirmed that the protein consists of six helices in solution and that the N-terminal arm is extremely flexible (Arrowsmith et al., 1990a, b;Lane, 1989). It has been postulated that this N-terminal arm may be involved in stabilising the DNA repressor complex by wrapping around the DNA as in the 1, repressors (Carey, 1989). More recently, this hypothesis has been questioned based on measurements of the effect of removing the first six residues on the affinity for operator DNA (Marmorstein et al., 1991).One of the first steps in probing the nature of the trprepressor -DNA interaction by NMR is the assignment of the _ _ _ _

Proton, carbon-13, and nitrogen-15 NMR backbone assignments and secondary structure of human interferon-.gamma.

“…indicates that spin diffusion must be extensive at the 70-ms mixing period used. However, both the number of NOE cross peaks and their relative intensities are comparable with those observed for other somewhat smaller proteins with longer NOE mixing times (Wüthrich et al, 1991;Borden et al, 1992;Marion et al, 1989a), and this degree of spin diffusion does not prohibit determination of secondary structure. For example, the stretch of </nn NOE connectivities from E10 through G19 observed in Figure 5 A (marked by dotted lines) contains part of the first -helix.…”

The interactions of Escherichia coli trp repressor with tryptophan and with an operator oligonucleotide

“…Resonance assignments are available for the repressortryptophan complex (Hyde et al, 1989(Hyde et al, ,1991Arrowsmith et al, 1990Czaplicki et al, 1991;Borden et al, 1991;and our unpublished results) and recently for the repressortryptophan-operator oligonucleotide complex (Zhang et al, 1994). These assignments have depended on extensive use of selective deuteration and general "N and "N/13C enrichment, and have led to determination of the solution structure (Zhao et al, 1993;., 1994).…”

Backbone Dynamics of trp Repressor Studied by 15N NMR Relaxation

“…Polynomial baseline corrections were applied in the F2 domain after Fourier transformation. Previously published amide proton/nitrogen assignments (Arrowsmith et al, 1990;Czaplicki et al, 1991;Borden et al, 1992;Ramesh et al, 1994) were used. Chemical shifts were measured relative to the Ala 29 resonance with 8.95 ppm in the 'H dimension and 126.90 ppm in the 15N dimension, while a total of 71 residues were assigned.…”