ATP-SENSITIVE TRYPTOPHANS OF HSP90

The nature of the interaction between the nucleotide ATP and hsp90 was investigated by observing fluorescence quenching of the four tryptoph an residues in hsp90 as a function of quencher type and temperature. A TP and acrylamide quench the fluorescence from tryptophan free in solu tion principally by static and collisional mechanisms, respectively. A crylamide quenching of tryptophan fluorescence in hsp90 is also princi pally collisional and identifies two classes of residues, one readily accessible to quenching the other less accessible. ATP quenching of tr yptophan fluorescence in hsp90 is more complex exhibiting no overall p referred mechanism. However, ATP competitively inhibits acrylamide que nching of the readily accessible class of tryptophan residues by stati c quenching with the quenching constant providing an upper limit for t he ATP dissociation constant. The ATP-free tryptophan dissociation con stant is more than a factor of three larger than that for ATP-hsp90 su ggesting that the ATP-hsp90 interaction is specific. The static quench ing of tryptophan fluorescence in hsp90 by ATP implies that the nucleo tide binds in close proximity to one or more of the tryptophan residue s. (C) 1998 Elsevier Science B.V. All rights reserved.