Neutron crystallographic analysis of the nucleotide-binding domain of Hsp72 in complex with ADP.

The 70 kDa heat-shock proteins (Hsp70s) are ATP-dependent molecular chaperones that contain an N-terminal nucleotide-binding domain (NBD) and a C-terminal substrate-binding domain. Hsp70s bind to misfolded/unfolded proteins and thereby prevent their aggregation. The ATP hydrolysis reaction in the NBD plays a key role in allosteric control of the binding of substrate proteins. In the present study, the neutron crystal structure of the NBD of Hsp72, a heat-inducible Hsp70 family member, was solved in complex with ADP in order to study the structure-function relationship with a focus on hydrogens. ADP bound to Hsp72 was fully deprotonated, and the catalytically important residues, including Asp10, Asp199 and Asp206, are also deprotonated. Neutron analysis also enabled the characterization of the water clusters in the NBD. Enzymatic assays and X-ray crystallographic analysis revealed that the Y149A mutation exhibited a higher ATPase activity and caused disruption of the water cluster and incorporation of an additional magnesium ion. Tyr149 was suggested to contribute to the low intrinsic ATPase activity and to stabilize the water cluster. Collectively, these structural studies will help to elucidate the molecular basis of the function of Hsp72.

Effects of the 2',4'-BNA modification on the sequence specificity of molecular beacons.

Molecular beacons (MBs) are stem-loop hairpin oligonucleotide probes with an internally quenched fluorophore. These probes recognize their targets with higher specificity than conventional linear probes. To further enhance sequence-specificity of MBs, we have designed and synthesized MBs having 2',4'-BNA modification. Thermodynamic analysis revealed that the MBs with the 2',4'-BNA-modified stem region have high sequence-specificity. In addition, the 2',4'-BNA modification in the loop region was also found to be efficient for discrimination of one base mismatch.