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. Author manuscript; available in PMC: 2006 Mar 21.

Published in final edited form as: Nature. 2005 Jun 9;435(7043):765–772. doi: 10.1038/nature03679

a) Amyloid core length and stability differ in fibres assembled at 4°C and 25°C. Acrylodan-labelled fibres assembled at 4°C () and 25°C () were denatured as in Fig. 1b. D_1/2 values were obtained from full GdmCl denaturation profiles (see Fig 1c).

(b) Intersubunit interfaces change in fibres assembled at different temperatures. Excimer fluorescence of pyrene-labelled mutants assembled in rotated unseeded reactions at 25°C (), 4°C ( or in seeded unrotated reactions with 4% seed formed at 4°C (). Pyrene fluorescence is similar in seeded and unseeded reactions.

(c) BMB cross-links at different positions bias assembly towards distinct prion strains. NM proteins cross-linked in denaturant were diluted into buffer, assembled into fibres, and used to transform [psi⁻] cells to [PSI⁺] state. Left, controls: uncross-linked WT fibres assembled at RT or at 4°C are biased towards the production of weak or strong strains, respectively²⁷. Soluble NM, buffer alone, and proteins cross-linked in the Central Core did not induce [PSI⁺] above background and were not scored. Values represent means ± SD (n=5 experiments)

(d) Strain biases created by cross-linking overcome the biases created by assembly at different temperatures. Whether they were assembled at 4°C or at RT, fibres cross-linked in the Head (G25C or G31C) produced mostly strong [PSI⁺] strains, and fibres cross-linked in the Tail (G96C and106C) produced mostly weak [PSI⁺] strains. Values represent means ± SD (n=5 experiments)

Inline graphic — a) Amyloid core length and stability differ in fibres assembled at 4°C and 25°C. Acrylodan-labelled fibres assembled at 4°C () and 25°C () were denatured as in Fig. 1b. D_1/2 values were obtained from full GdmCl denaturation profiles (see Fig 1c).

(b) Intersubunit interfaces change in fibres assembled at different temperatures. Excimer fluorescence of pyrene-labelled mutants assembled in rotated unseeded reactions at 25°C (), 4°C ( or in seeded unrotated reactions with 4% seed formed at 4°C (). Pyrene fluorescence is similar in seeded and unseeded reactions.

(c) BMB cross-links at different positions bias assembly towards distinct prion strains. NM proteins cross-linked in denaturant were diluted into buffer, assembled into fibres, and used to transform [psi⁻] cells to [PSI⁺] state. Left, controls: uncross-linked WT fibres assembled at RT or at 4°C are biased towards the production of weak or strong strains, respectively²⁷. Soluble NM, buffer alone, and proteins cross-linked in the Central Core did not induce [PSI⁺] above background and were not scored. Values represent means ± SD (n=5 experiments)

(d) Strain biases created by cross-linking overcome the biases created by assembly at different temperatures. Whether they were assembled at 4°C or at RT, fibres cross-linked in the Head (G25C or G31C) produced mostly strong [PSI⁺] strains, and fibres cross-linked in the Tail (G96C and106C) produced mostly weak [PSI⁺] strains. Values represent means ± SD (n=5 experiments)