RESUMO
The nature of the interaction between the nucleotide ATP and hsp90 was investigated by observing fluorescence quenching of the four tryptophan residues in hsp90 as a function of quencher type and temperature. ATP and acrylamide quench the fluorescence from tryptophan free in solution principally by static and collisional mechanisms, respectively. Acrylamide quenching of tryptophan fluorescence in hsp90 is also principally collisional and identifies two classes of residues, one readily accessible to quenching the other less accessible. ATP quenching of tryptophan fluorescence in hsp90 is more complex exhibiting no overall preferred mechanism. However, ATP competitively inhibits acrylamide quenching of the readily accessible class of tryptophan residues by static quenching with the quenching constant providing an upper limit for the ATP dissociation constant. The ATP-free tryptophan dissociation constant is more than a factor of three larger than that for ATP-hsp90 suggesting that the ATP-hsp90 interaction is specific. The static quenching of tryptophan fluorescence in hsp90 by ATP implies that the nucleotide binds in close proximity to one or more of the tryptophan residues.
