Abstract
2'-Deoxy-3'-anthraniloyl adenosine-5-triphosphate (ANT-dATP) coordinated to Tb3+ was used as an environmentally sensitive probe of the nucleotide-binding site of GroEL. Tb3+.ANT-dATP recognizes the nucleotide-binding site of GroEL and inhibits ATPase activity. Sensitized luminescence, arising from resonance energy transfer from the anthraniloyl moiety to Tb3+, is substantially enhanced in the presence of GroEL. Binding of denatured mitochondrial malate dehydrogenase to the apical domain of GroEL causes a red shift in the fluorescence emitted by anthraniloyl and further enhancement in the phosphorescence emitted by Tb3+ upon excitation at 320 nm. It is suggested that binding of the protein substrate initiates domain movement, which is extended to the nucleotide-binding site. The luminescence results are discussed in reference to the structure of GroEL derived from x-ray crystallographic studies.
Highlights
2*-Deoxy-3*-anthraniloyl adenosine-5-triphosphate (ANT-dATP) coordinated to Tb31 was used as an environmentally sensitive probe of the nucleotide-binding site of GroEL
Binding of denatured mitochondrial malate dehydrogenase to the apical domain of GroEL causes a red shift in the fluorescence emitted by anthraniloyl and further enhancement in the phosphorescence emitted by Tb31 upon excitation at 320 nm
It is suggested that binding of the protein substrate initiates domain movement, which is extended to the nucleotide-binding site
Summary
2*-Deoxy-3*-anthraniloyl adenosine-5-triphosphate (ANT-dATP) coordinated to Tb31 was used as an environmentally sensitive probe of the nucleotide-binding site of GroEL. Several lines of experimental evidence suggest that binding of nucleotides to GroEL induces conformational changes in the apical domain, which influences the affinity of peptides and unfolded proteins (4 – 6). ATPase activity(4), it is not known whether the same binding process is responsible for conformational changes at the level of the nucleotide-binding site in the equatorial domain. In an attempt to detect coupling between the apical and equatorial domains of GroEL, the luminescence properties of a probe coordinated to the nucleotide-binding site were investigated in detail. The results obtained with two different spectroscopic methods, i.e. fluorescence and phosphorescence, covering the time range 2 ns to 1ms, lend support to the contention that the interaction of unfolded proteins with GroEL perturbs the microenvironment of the nucleotide site
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