These studies revealed that the cytosolic region can bind to Diva and that, at the structural level, is largely disordered with residual a-helical conformation at a population of ,13% . Hrk is therefore a potential model example combining both intrinsic disorder and membrane binding capabilities. Up to date high-resolution structural studies on BH3-only buy MK-2206 proteins in isolation are lacking except for Bid , the only known member with a defined fold. In an effort to improve our knowledge on the operating mode of BH3-only proteins we have characterized Hrk by NMR and circular dichroism to obtain atomic-detailed information on the conformational preferences of the cytosolic and TM domains. We have used synthetic constructs that encompass: 1) the entire cytosolic domain, 2) a smaller fragment including the BH3 motif and short flanking sequences at its N- and C-termini, 3) the TM domain. Firstly, we show using enzyme immunoassays and NMR that the cytosolic constructs are capable of binding Bcl-2 and Bcl-xL, the two proteins known to be implicated in Hrk��s apoptotic function . In addition, by combining previous structural data on the entire cytosolic domain and new data on the smaller construct in water and wateralcohol mixture we find that a-helical propensity is confined in a 25-residue long fragment that MDV3100 chemical information comprises the 15 amino acid long BH3 motif. We have determined the 3D structure of the shorter cytosolic construct, revealing that it is strikingly similar to the structure of BH3-peptides bound to prosurvival proteins. This result suggests that intrinsic residual structure in disordered BH3- only proteins could play a fundamental role in the binding mechanism, as preformed conformations involving the BH3 domain are likely implicated in the interaction with survival partners. Furthermore, we found that the TM domain of Hrk is poorly soluble in aqueous solution albeit it readily inserts into micelles. This result agrees with the reported localization of Hrk in membranes of intracellular organelles , suggesting that membrane binding is an important determinant of Hrk��s function. We also show that the TM domain is monomeric in the presence of micelles, which points to a role as membrane anchor, in contrast to the TM domain of BNip3 found to function as a membrane permeabilization device . Finally, we report the 3D structure of Hrk��s TM domain in micelles revealing that it forms an a-helix followed by a well-ordered turn. The length of the helix and turn is ,30A�� , which is similar to the thickness of cellular membranes.