RESUMEN

We present an NMR investigation of the nucleotide-dependent conformational properties of a 44-kDa nucleotide binding domain (NBD) of an Hsp70 protein. Conformational changes driven by ATP binding and hydrolysis in the N-terminal NBD are believed to allosterically regulate substrate affinity in the C-terminal substrate binding domain. Several crystal structures of Hsc70 NBDs in different nucleotide states have, however, not shown significant structural differences. We have previously reported the NMR assignments of the backbone resonances of the NBD of the bacterial Hsp70 homologue Thermus thermophilus DnaK in the ADP-bound state. In this study we show, by assigning the NBD with the ATP/transition state analogue, ADP.AlFx, bound, that it closely mimics the ATP-bound state. Chemical shift difference mapping of the two nucleotide states identified differences in a cluster of residues at the interface between subdomains 1A and 1B. Further analysis of the spectra revealed that the ATP state exhibited a single conformation, whereas the ADP state was in slow conformational exchange between a form similar to the ATP state and another state unique to the ADP-bound form. A model is proposed of the allosteric mechanism based on the nucleotide state altering the balance of a dynamic equilibrium between the open and closed states. The observed chemical shift perturbations were concentrated in an area close to a previously described J-domain binding channel, confirming the importance of that region in the allosteric mechanism.

Asunto(s)

Proteínas Bacterianas/química , Proteínas HSP70 de Choque Térmico/química , Proteínas HSP70 de Choque Térmico/metabolismo , Espectroscopía de Resonancia Magnética , Chaperonas Moleculares/química , Nucleótidos/metabolismo , Thermus thermophilus/química , Adenosina Difosfato/metabolismo , Adenosina Trifosfato/metabolismo , Regulación Alostérica , Secuencia de Aminoácidos , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Sitios de Unión , Clonación Molecular , Escherichia coli/genética , Expresión Génica , Proteínas HSP70 de Choque Térmico/genética , Modelos Moleculares , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Datos de Secuencia Molecular , Conformación Proteica , Relación Estructura-Actividad , Especificidad por Sustrato

RESUMEN

Two new ruthenium(II) polypyridyl dimers containing the large planar aromatic bridging ligands 9,11,20,22-tetraazatetrapyrido[3,2-a:2'3'-c:3' ',2''-l:2''',3'''-n]pentacene (tatpp) and 9,11,20,22-tetraazatetrapyrido[3,2-a:2'3'-c:3'',2' '-l:2''',3'''-n]pentacene-10,21-quinone (tatpq) have been synthesized and characterized by (1)H and (13)C NMR, MALDI mass spectrometry, and elemental analyses. The electronic properties (UV-vis, redox, photophysical) of these dimers are analyzed in the context of orbital calculations (PM3 level) on the bridging ligands. A localized orbital model is proposed in which low-lying acceptor orbitals on the center portion of the ligands effectively quench the Ru(II)-based MLCT emission via a mechanism that can be viewed as intramolecular electron transfer to specific subunits of the bridges.