Heme as an optical probe of a conformational transition of ovine recPrP.
The prion protein occurs as a globular domain and a leading fragment whose structure is not well-defined. For the ovine species, all of the tryptophan residues are in the initial fragment, while the globular domain is rich in tyrosine residues. Using heme as a spectroscopic probe, we have studied the recombinant prion protein before and after a temperature-induced conformational change. As for most heme proteins, the absorption spectrum of heme-CO displays a red shift upon binding to the protein, and both the Y and W fluorescence are highly quenched. Flash photolysis kinetics of the PrP-heme-CO complex shows a low yield for the bimolecular phase, indicating a pocket around the hemes. By comparing the holoprotein and the truncated sequence corresponding to the globular domain, the stoichiometry was determined to be five hemes for the globular domain and two hemes for the leading fragment. At high temperature, the hemes are released; upon cooling, only two hemes bind, and only the tryptophan fluorescence is quenched; this would indicate that the globular domain has formed a more compact structure, which is inert with respect to the hydrophobic probe. The final state of polymerization is perturbed if the synthetic peptide "N3" (PrP residues 142-166, which include the first helix) is added to the prion protein solution; the temperature cycle no longer reduces the number of heme binding sites. This would indicate that the peptide may alter or inhibit the polymer formation.
Pato
Christine
C
INSERM, U473, 84 rue du Général Leclerc, 94276 Le Kremlin Bicêtre Cedex, France.
Célier
Chantal
C
Rezaei
Human
H
Grosclaude
Jeanne
J
Marden
Michael C
MC
D000818:Animals; D002248:Carbon Monoxide / Q000378:metabolism; D005456:Fluorescent Dyes; D006418:Heme; D010455:Peptides / Q000737:chemistry*; D011328:Prions / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012756:Sheep; D013050:Spectrometry, Fluorescence; D013696:Temperature; D014364:Tryptophan / Q000737:chemistry; D014443:Tyrosine / Q000737:chemistry
D005456:Fluorescent Dyes; D010455:Peptides; D011328:Prions; D011994:Recombinant Proteins; D014443:Tyrosine; D006418:Heme; D002248:Carbon Monoxide; D014364:Tryptophan