DETECTION OF TRANSMISSIBLE SPONGIFORM ENCEPHALOPATHY AGENTS IN LIVESTOCK, WILDLIFE, AGRICULTURAL PRODUCTS, AND THE ENVIRONMENT
Location: Foodborne Contaminants Research
Title: Probing structural differences between PrPC and PrPSc by surface nitration and acetylation: evidence of conformational change in the C-terminus
| Gong, Binbin - |
| Ramos, Adriana - |
| Vazquez-Fernandez, Ester - |
| Alonso, Jana - |
| Requena, Jesus - |
Submitted to: Biochemistry
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: April 25, 2011
Publication Date: April 28, 2011
Citation: Gong, B., Ramos, A., Vazquez-Fernandez, E., Silva, C.J., Alonso, J., Requena, J.R. 2011. Probing structural differences between PrPC and PrPSc by surface nitration and acetylation: evidence of conformational change in the C-terminus. Biochemistry. 50:4963-4972.
Interpretive Summary: Two chemicals (tetranitromethane (TNM) and acetic anhydride) were used to react with a model prion protein (Syrian hamster recombinant PrP(90-231) (rPrP)) and an infectious prion (PrP27-30). This was done to identify the structural differences between the two proteins. After the chemical reacted with the proteins, the chemically modified proteins were suitably prepared for chemical analysis using mass spectrometry (Matrix-Assisted Laser Desorption/Ionization-Time-Of Flight (MALDI-TOF)). Even though rPrP and PrP 27-30 were reacted with the same reagents, they reacted differently, based on their structures. The TNM reagent reacted with some parts of rPrP (C-terminus; E221-R229 and H111-R136) and not with those same parts of PrP 27-30. The TNM reagent reacted with parts of PrP 27-30 (Y149-R151, Y157-R164, and R151-Y162) and not with those same parts of rPrP. Acetic acid reacted with the same part (G90-K106) of both rPrP and PrP 27-30. These results suggest that there are important conformational differences in the C-terminal regions of rPrP and PrP 27-30. In addition the amino acids (tyrosines) contained in one region (Y149 to R164) are more accessible in PrP 27-30 than in rPrP. Lastly, the amino terminal region of PrP 27-30 and rPrP react equally well with these reagents. These data should help validate and construct structural models of prions.
We used two chemical modifiers, tetranitromethane (TNM) and acetic anhydride, which specifically target accessible tyrosine and lysine residues, respectively, to modify Syrian hamster recombinant PrP(90-231) (rPrP) and PrP27-30, aiming at finding locations of conformational change. Modified proteins were subjected to in-gel proteolytic digestion with trypsin or chymotrypsin, and subsequent analysis by mass spectrometry (MALDI-TOF). Several differences in chemical reactivity were observed. With TNM, the most conspicuous reactivity difference seen involves peptide E221-R229 (containing Y226 and Y227) which in rPrP was much more extensively modified than in PrP27-30; peptide H111-R136, containing Y128, was also more modified in rPrP. Conversely, peptides Y149-R151, Y157-R164, and R151-Y162 suffered more extensive modification in PrP27-30. Acetic anhydride modified very extensively peptide G90-K106, containing K101, K104, K106 and the amino terminus, in both rPrP and PrP27-30. These results suggest that 1) PrPSc exhibits important conformational differences in the C-terminal region with respect to PrPC, resulting in loss of solvent accessibility of Y225 and Y226, very solvent-exposed in the latter conformation; since other results suggest preservation of the two C-terminal helices, this might mean that these are tightly packed in PrPSc; 2) contrarily, tyrosines contained in the stretch spanning approximately from Y149 to R164 are more accessible in PrPSc, suggesting rearrangements in Alpha-helix H1 and the short Beta-sheet of PrPC; 3) the amino terminal region of PrPSc is very accessible. These data should help validate and construct structural models of PrPSc.