Abstract
2'-Deoxy-3'-anthraniloyladenosine-5-triphosphate(Ant-dATP) was used as an environmentally sensitive probe of the nucleotide-binding site of the molecular chaperone Hsc70. When coordinated to the lanthanide ion Tb3+, Ant-dATP is not hydrolyzed by Hsc70. The lanthanide ion acts as strong competitive inhibitor with respect to Mg2+ (Ki = 0.1 microM). Tb.Ant-dATP recognizes the nucleotide site of Hsc70 as revealed by an increase in the emission anisotropy from 0.03 to 0.21 and by a change in the fluorescence-decay time from 2.52 ns to 3.75 ns. Sensitized luminescence arising from resonance energy transfer from the anthraniloyl group to Tb3+ is substantially enhanced in the presence of Hsc70. Binding of a 20-amino-acid-residue peptide (Rnase-S peptide) to Hsc70 causes a blue shift in the fluorescence spectrum of Ant-dATP and enhances Tb3+ luminescence upon excitation at 330 nm. It is postulated that binding of the peptide to the COOH-terminal domain of Hsc70 initiates domain movement and the structural changes might extend to the nucleotide-binding site.
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