Amino terminal interaction in the prion protein identified using fusion to green fluorescent protein.
In contrast to the well-characterized carboxyl domain, the amino terminal half of the mature cellular prion protein has no defined structure. Here, following fusion of mouse prion protein fragments to green fluorescence protein as a reporter of protein stability, we report extreme variability in fluorescence level that is dependent on the prion fragment expressed. In particular, exposure of the extreme amino terminus in the context of a truncated prion protein molecule led to rapid degradation, whereas the loss of only six amino terminal residues rescued high level fluorescence. Study of the precise endpoints and residue identity associated with high fluorescence suggested a domain within the amino terminal half of the molecule defined by a long-range intramolecular interaction between 23KKRPKP28 and 143DWED146 and dependent upon the anti-parallel beta-sheet ending at residue 169 and normally associated with the structurally defined carboxyl terminal domain. This previously unreported interaction may be significant for understanding prion bioactivity and for structural studies aimed at the complete prion structure.
Yao
Yongxiu
Y
School of Animal and Microbial Sciences, The University of Reading, Reading RG6 6AJ, UK.
Ren
Junyuan
J
Jones
Ian M
IM
D000818:Animals; D002460:Cell Line; D004591:Electrophoresis, Polyacrylamide Gel; D015870:Gene Expression; D018014:Gene Transfer Techniques; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D016297:Mutagenesis, Site-Directed; D018621:PrPC Proteins / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding / Q000235:genetics / Q000502:physiology; D011487:Protein Conformation; D017510:Protein Folding; D017434:Protein Structure, Tertiary / Q000502:physiology; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism*; D018411:Spodoptera
D008164:Luminescent Proteins; D018621:PrPC Proteins; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins