Approximate three-fold excess of p300 TAZ2 to MedChemExpress BTZ-043 samples of the BMyb TAD resulted in a shift in the tryptophan fluorescence maximum from 354 to 344 nm, as shown in figure 2B, which clearly reflects a change in the tryptophan environment on formation of the B-Myb TAD-TAZ2 complex. This also suggests that the region encompassing one or both tryptophan residues in the B-Myb TAD adopts a folded conformation on binding to the TAZ2 domain. Unfortunately, given the low extinction coefficient of p300 TAZ2 (,1490 M21 cm21) and the required presence of DTT in the buffers it was not possible to accurately determine the protein concentration of TAZ2 [49]. This precludes the possibilityof using fluorescence titration data to reliably determine the affinity or stoichiometry of the complex. To confirm the specificity of the B-Myb TAD-p300 TAZ2 interaction, and to identify the residues of TAZ2 involved in interactions with the B-Myb TAD, NMR spectroscopy was used to monitor changes in the backbone amide signals of p300 TAZ2 induced by complex formation. Figure 5A shows typical 15N/1H HSQC spectra obtained from samples of 15N-labelled p300 TAZ2 (100 mM) in the absence (red) and presence (black) of an equivalent amount of unlabelled B-Myb TAD. The addition of the B-Myb TAD results in significant shifts in the positions of a subset of signals, as well as substantial line broadening leading to a loss of a few peaks. Addition of a second molar equivalent of B-Myb TAD resulted in further line broadening and loss of the majority of the peaks (data not shown). The extent of the line broadening observed required acquisition times of about 12 hours to obtainFeatures of the B-Myb TAD-p300 TAZ2 Complexcould not be determined due to missing backbone amide resonances in 15N/1H HSQC spectrum of the complex.Discussion B-Myb TADPrevious reports have identified the poorly characterised, central transactivation region of B-Myb as the binding site for several functional partner proteins [15], [50]. We have expressed the region corresponding to the B-Myb transactivation domain (residues 275?76) in E. coli as a GST fusion protein and characterised the properties of the purified B-Myb TAD using a range of spectroscopic techniques. CD and NMR spectra of the BMyb TAD clearly show 24195657 that it forms a random coil polypeptide, with no MedChemExpress Naringin regular secondary or tertiary structure. This is consistent with the observed tryptophan fluorescence emission maximum of 354 nm, which indicates that the two tryptophan side chains are fully exposed to the aqueous environment. The random coil nature of the B-Myb TAD is not entirely unexpected, as this region contains a fairly high proportion of polar and charged amino acid residues (Gln/Asn 10 , Ser/Thr 15 , Asp/Glu 18 , Lys/Arg 6 ), as well as many proline residues (11 ), which are features associated with intrinsically disordered regions and are characteristics of many transcriptional activation domains [51], [52]. Unstructured TADs have been reported for a number of transcription factors, including the kinase-inducible activation domain (KID) of CREB [53], the Cterminal activation domain of Hif-1a [54], [55], the activation domains of STAT-1 and 2 [56] and the activation domain of the glucocorticord receptor [57]. Many transcriptional regulators are known to contain similar unstructured regions that adopt well defined conformations on binding to functional partner proteins [32], [54], [56], [58], [59], [60], [61]. The intrinsically disordered nature o.Approximate three-fold excess of p300 TAZ2 to samples of the BMyb TAD resulted in a shift in the tryptophan fluorescence maximum from 354 to 344 nm, as shown in figure 2B, which clearly reflects a change in the tryptophan environment on formation of the B-Myb TAD-TAZ2 complex. This also suggests that the region encompassing one or both tryptophan residues in the B-Myb TAD adopts a folded conformation on binding to the TAZ2 domain. Unfortunately, given the low extinction coefficient of p300 TAZ2 (,1490 M21 cm21) and the required presence of DTT in the buffers it was not possible to accurately determine the protein concentration of TAZ2 [49]. This precludes the possibilityof using fluorescence titration data to reliably determine the affinity or stoichiometry of the complex. To confirm the specificity of the B-Myb TAD-p300 TAZ2 interaction, and to identify the residues of TAZ2 involved in interactions with the B-Myb TAD, NMR spectroscopy was used to monitor changes in the backbone amide signals of p300 TAZ2 induced by complex formation. Figure 5A shows typical 15N/1H HSQC spectra obtained from samples of 15N-labelled p300 TAZ2 (100 mM) in the absence (red) and presence (black) of an equivalent amount of unlabelled B-Myb TAD. The addition of the B-Myb TAD results in significant shifts in the positions of a subset of signals, as well as substantial line broadening leading to a loss of a few peaks. Addition of a second molar equivalent of B-Myb TAD resulted in further line broadening and loss of the majority of the peaks (data not shown). The extent of the line broadening observed required acquisition times of about 12 hours to obtainFeatures of the B-Myb TAD-p300 TAZ2 Complexcould not be determined due to missing backbone amide resonances in 15N/1H HSQC spectrum of the complex.Discussion B-Myb TADPrevious reports have identified the poorly characterised, central transactivation region of B-Myb as the binding site for several functional partner proteins [15], [50]. We have expressed the region corresponding to the B-Myb transactivation domain (residues 275?76) in E. coli as a GST fusion protein and characterised the properties of the purified B-Myb TAD using a range of spectroscopic techniques. CD and NMR spectra of the BMyb TAD clearly show 24195657 that it forms a random coil polypeptide, with no regular secondary or tertiary structure. This is consistent with the observed tryptophan fluorescence emission maximum of 354 nm, which indicates that the two tryptophan side chains are fully exposed to the aqueous environment. The random coil nature of the B-Myb TAD is not entirely unexpected, as this region contains a fairly high proportion of polar and charged amino acid residues (Gln/Asn 10 , Ser/Thr 15 , Asp/Glu 18 , Lys/Arg 6 ), as well as many proline residues (11 ), which are features associated with intrinsically disordered regions and are characteristics of many transcriptional activation domains [51], [52]. Unstructured TADs have been reported for a number of transcription factors, including the kinase-inducible activation domain (KID) of CREB [53], the Cterminal activation domain of Hif-1a [54], [55], the activation domains of STAT-1 and 2 [56] and the activation domain of the glucocorticord receptor [57]. Many transcriptional regulators are known to contain similar unstructured regions that adopt well defined conformations on binding to functional partner proteins [32], [54], [56], [58], [59], [60], [61]. The intrinsically disordered nature o.
