Amino Acid Proximities in Two Sup35 Prion Strains Revealed by Chemical Cross-Linking [Microbiology]

August 11th, 2015 by Wong, S.-H., King, C.-Y.

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 (Met) substitutions and two alanine (Ala) 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 inter-molecular cross-linking were distinguished by tandem mass spectrometry analysis on fibers seeded from solutions containing equal amount of 14N and 15N-labeled protein. Fibers propagating the VL strain type exhibited intra-molecular cross-linking between amino acid residues 3 and 28, as well as intra- and inter-molecular linking between 32 and 55. Inter- and intra-molecular 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.