Amino Acid Proximities in Two Sup35 Prion Strains Revealed by Chemical Cross-linking

Area of Science:

• Biochemistry
• Structural Biology
• Yeast Genetics

Background:

• The yeast prion [PSI] involves different Sup35 protein folding patterns forming amyloid fibers.
• Understanding these distinct structures is key to prion propagation mechanisms.

Purpose of the Study:

• To investigate the structural differences between yeast prion [PSI] strains using chemical cross-linking.
• To map residue-residue proximities within Sup35 amyloid fibers of different strains.

Main Methods:

• Biosynthetic incorporation of photo-reactive cross-linker p-benzoyl-l-phenylalanine (pBpa) into Sup35 fragments.
• Mass spectrometry analysis of cross-linked Sup35 amyloid fibers from VK and VL prion strains.
• Utilizing isotopic labeling ((14)N/(15)N) to distinguish intra- and intermolecular cross-links.

Main Results:

• Specific residue adjacencies were identified in VL strain fibers (3-28, 32-55) and VK strain fibers (32-55).
• These cross-linking patterns confirmed distinct structural conformations for VK and VL prion strains.
• The engineered Sup35 fragments faithfully transmitted strain characteristics.

Conclusions:

• Cross-linking provides insights into the spatial arrangement of amino acid residues in different [PSI] strains.
• Structural variations in Sup35 fibers underlie the distinct characteristics of yeast prion strains.
• This approach can constrain models of prion fiber folding.