Binding of a fluorescent nucleotide analog to Hsc70. The effect of peptide protein interactions on the luminescence properties of the probe.
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.
Churchich
J E
JE
Department of Biochemistry, University of Tennessee, Knoxville, USA.
D000255:Adenosine Triphosphate / Q000031:analogs & derivatives* / Q000378:metabolism; D000818:Animals; D002352:Carrier Proteins / Q000378:metabolism*; D002417:Cattle; D005456:Fluorescent Dyes / Q000378:metabolism*; D050883:HSC70 Heat-Shock Proteins; D018840:HSP70 Heat-Shock Proteins; D010455:Peptides / Q000378:metabolism; D011485:Protein Binding; D012260:Ribonucleases / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D062367:ortho-Aminobenzoates / Q000378:metabolism*
D002352:Carrier Proteins; D005456:Fluorescent Dyes; D050883:HSC70 Heat-Shock Proteins; D018840:HSP70 Heat-Shock Proteins; D010455:Peptides; D062367:ortho-Aminobenzoates; C066294:3'-anthraniloyl-2'-deoxy-ATP; D000255:Adenosine Triphosphate; D012260:Ribonucleases; C022542:ribonuclease S