The binding between Bcl-xL and Hrk was also studied by NMR. The 15N-HSQC spectrum of unbound Bcl-xL changes significantly by adding Hrk-22_53 at equimolar amount indicating direct interaction . An analogous result is obtained by the addition of Hrk-DTM . Moreover, the titration of Bcl-xL with either Hrk-22_53 or Hrk-DTM leads to almost identical changes in the NMR spectra suggesting that the interaction is similar at the structural level. This behavior was also order ICG-001 observed for the Diva/Hrk complexes mentioned above . By increasing the concentration of the Hrk fragments in excess relative to Bcl-xL, no additional changes in the spectra were observed. This result indicates binding saturation at equimolar amounts of Bcl-xL and the Hrk constructs, and the formation of 1:1 Bcl-xL/Hrk-22_53 and Bcl-xL/Hrk-DTM complexes. Furthermore, two sets of NMR signals are present in the spectra of Bcl-xL/Hrk mixtures at 1:0.3 molar ratio. One set corresponds to the spectrum of free Bcl-xL and the second set to the complex, as inferred from signal overlap of the spectra at 1:0.3 molar ratio with both the spectra of free BclxL and the one resulting from the equimolar mixture . This result indicates that exchange is in the slow regime relative to the chemical shift time scale. Binding saturation and slow exchange suggest high affinity constants for these complexes, in agreement with previous fluorescence data reporting a dissociation constant value of 92 nM for a complex between Bcl-xL and a short 20 residue-long peptide comprising the BH3 domain of Hrk . Nevertheless, further studies are necessary to understand the structural, dynamics and energetic factors implicated in the BclxL/ Hrk interaction. Previous structural data from our group show that the cytosolic domain of Hrk is largely disordered . In particular, small dispersion of amide 1H chemical shifts and NMR signals associated to methyl groups indicate that the protein is unfolded and lacks a hydrophobic core . However, previous CD data on Hrk-DTM indicate the formation of approximately 13% population of a-helical structure. This population increases to 35% upon TFE addition at 35% , a solvent known to promote secondary structure formation in protein fragments and peptides with intrinsic conformational propensity . To further characterize structurally Hrk-DTM by NMR is necessary to Abmole Company LDK378 reduce signal overlap originating from both the small chemical shift dispersion resulting from the low structure population and the numerous signals of the rather long protein fragment.