Diapositiva 1

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Amyloid Fibrils formation and Amorphous
Aggregation in Concanavalin A
Fabio Librizzi Dipartimento di Scienze Fisiche e
Astronomiche Università di Palermo
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Concanavalin A

• MW27.000
• b-structure, no a-helices
• Jelly roll fold
• 4 tryptophans
• No disulphide bonds
• Isoelectric point pH5

• 1 Ca2 ed 1Mn2 in each subunit
• Bind carbohydrates reversibly and specifically

• Protein-Carbohydrate interactions
• Medical applications (Liver injury)

pH 6,5
Tetramer
dimer
D.F. Senear Biochemistry 1981 Emsley Nature
1994, L. Bhattacharyya J. Biol. Chem 1991 Mitra
Biochemistry 2002 Sinha Biophys. J. 2005
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Con A induces Programmed Cell Death on cortical
neurons
Neuron Coltures exposed to 50nm ConA 24h
The suggested mechanism involves the
cross-linking of cell surface receptors,
associated with the clustering of protein
molecules on the surface.
a-b) Control neurons c-d) Neurons treated with
ConA for 24 h
Cribbs et al. Neuroscience 1996 Anderson et
al., J. Neurochem 1995
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Thermal Induced Aggregation
Atomic Force Microscopy
Scattering
Circular Dichroism
Thioflavin T ANS Tryptophans
Morphology of aggregates
Formation of large objects
Secondary structure conformational changes
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Thioflavin T fluorescence
Scattering
pH
Different aggregation pathways Amyloid
formation (high pH) Amorphous aggregation (low pH)
Con A 0.5mg/ml TT, 40C lexc440nm
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AFM measurements
pH 5 after 40 min at 40C
pH 9 after 40 min at 40C
Scan size 3 mm Z-range 15 nm
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ANS emission kinetic
Thioflavin T
pH
creation and evolution of new hydrophobic regions
Con A 0.5mg/ml -ANS at T40C lexc380nm
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Circular Dichroism
Thioflavin T
Time evolution of CD signal at 210nm
pH
pH
Fibril formation involves Secondary structure
conformational changes
Con A 0.5mg/ml pH9 40C.
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Up to now...

• the aggregation process of ConA can evolve
  through two distinct pathways, leading to the
  formation of amorphous aggregates (low pH) or
  amyloid fibrils (high pH).
• the formation of amyloid fibrils requires large
  conformational changes of the protein, both at
  the secondary and tertiary structural level.
• No lag phase, even at much lower protein
  concentration, down to 0.05 mg/ml. Weak
  concentration dependence. Probably, the
  fibrillation process is not affected by any kind
  of nucleation neither by cooperative mechanisms.

• Mechanisms of fibril formation
• Monomerization
• Neuronal Cell death

?
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Fibril formation at pH 7, TT-fluorescence
Succinyl-Con A
T 37 C ? Con A ? Succinyl-Con A
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Thermal aggregation of Succinyl-Con A
Fibril formation at high pH (slower than Con
A) Conformational changes at secondary and
tertiary structure level
pH 9
Temperature scan, 12C/h
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Succinyl-Con A Trp-fluorescence pH 9, 40C
Intensity
Frequency
Bandwidth Conformational heterogeneity
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People...
Università di Palermo, DSFA Valeria Vetri Fabio
Librizzi Valeria Militello Maurizio
Leone Università di Genova, Dip. di
Fisica Claudio Canale Annalisa Relini Alessandra
Gliozzi
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S-Con A
Con A