Amino Acid Proximities in Two Sup35 Prion Strains Revealed by Chemical Cross-linking.
Strains of the yeast prion [PSI] are different folding patterns of the same Sup35 protein, which stacks up periodically to form a prion fiber. Chemical cross-linking is employed here to probe different fiber structures assembled with a mutant Sup35 fragment. The photo-reactive cross-linker, p-benzoyl-l-phenylalanine (pBpa), was biosynthetically incorporated into bacterially prepared recombinant Sup(1-61)-GFP, containing the first 61 residues of Sup35, followed by the green fluorescent protein. Four methionine substitutions and two alanine substitutions were introduced at fixed positions in Sup(1-61) to allow cyanogen bromide cleavage to facilitate subsequent mass spectrometry analysis. Amyloid fibers of pBpa and Met/Ala-substituted Sup(1-61)-GFP were nucleated from purified yeast prion particles of two different strains, namely VK and VL, and shown to faithfully transmit specific strain characteristics to yeast expressing the wild type Sup35 protein. Intra- and intermolecular cross-linking were distinguished by tandem mass spectrometry analysis on fibers seeded from solutions containing equal amounts of (14)N- and (15)N-labeled protein. Fibers propagating the VL strain type exhibited intra- and intermolecular cross-linking between amino acid residues 3 and 28, as well as intra- and intermolecular linking between 32 and 55. Inter- and intramolecular cross-linking between residues 32 and 55 were detected in fibers propagating the VK strain type. Adjacencies of amino acid residues in space revealed by cross-linking were used to constrain possible chain folds of different [PSI] strains.
From the Molecular Cell Biology, Taiwan International Graduate Program, Academia Sinica and Graduate Institute of Life Sciences, National Defense Medical Center, Taipei 114, Taiwan and the Institute of Molecular Biology, Academia Sinica, Taipei 11529, Taiwan.
Shenq-Huey
SH
Wong
the Institute of Molecular Biology, Academia Sinica, Taipei 11529, Taiwan cking@imb.sinica.edu.tw.
Chih-Yen
CY
King
D000595:Amino Acid Sequence; D000596:Amino Acids; D003432:Cross-Linking Reagents / Q000494:pharmacology*; D008969:Molecular Sequence Data; D010454:Peptide Termination Factors / Q000737:chemistry*; D012441:Saccharomyces cerevisiae; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry*; D014466:Ultraviolet Rays
D000596:Amino Acids; D003432:Cross-Linking Reagents; D010454:Peptide Termination Factors; C068068:SUP35 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins
Sup35; [PSI]; amyloid; chemical biology; mass spectrometry (MS); prion strain; protein cross-linking; yeast