urop presentation
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Fluorescent Dye Interaction with Flemish Mutation in Alzheimer’s Disease
Stephanie LeNowick Lab
May 14, 2016
Overview
• Mutations in Alzheimer’s disease• Flemish Mutation–ThioflavinT co-crystallization–Congo Red co-crystallization
Aβ15-23
KMDAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVVIAAβ1-42
AβAPP
Aβ15-23
KMDAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVVIA
Aβ1-42
Q K L V F F A E DAβ15-23
--KQ
NOsaka E22 DeletionItalian E22KDutch E22QFlemish A21G
Iowa D23N
G
AβAPP
A21G
Alanine Glycine
Flemish SHao
Q K L V F F A E D
D E A F F V L K Q
Flemish SHao
Hao
Phenyalanine with Iodide A21G
Ornithine
Fibril Structure of Flemish Mutation
Fluorescent Dyes
• Why are dyes used?• Provides insight on Alzheimer’s Disease
Thioflavin T (ThT) co-crystallization
• Structure of Tht
Crystal Screening Tray Condition
• C1 well solution (based off KAC240)– 0.2 M NaCitrate (no pH)– 0.1 M Hepes Na pH 7.0– 35% MPD
• Vary concentrations of ThT
Control 5.6 mM 4.0 mM 3.2 mM 2.4 mM 1.6mM
650 µL C1350 µL water
650 µL C1350 µL of 16
mM ThT
650 µL C1250 µL 16 mM ThT
100 µL water
650 µL C1200 µL 16 mM ThT
150 µL water
650 µL C1150 µL 16 mM ThT
200 µL water
650 µL C1100 µL ThT 16 mM ThT
250 µL water
0.8 mM 0.4 mM 0.04 mM 0.004 mM 0.0004 mM650 µL C1
50 µL 16 mM ThT
300 µL water
650 µL C1100 µL 4 mM
ThT250 µL water
650 µL C1100 µL 0.4 mM ThT
250 µL water
650 µL C110 µL 0.4 mM ThT
340 µL water
650 µL C11 µL 0.04 mM ThT
349 µL water
A3 Well 650 µL C1 250 µL 16 mM ThT 100 µL water
Control Well 650 µL C1 350 µL water
0 mM ThT 4.0 mM ThT
Peptide #2
Peptide #1
Interaction
Congo Red
• Structure of Congo Red
Control 5.6 mM 4.0 mM 3.2 mM 2.4 mM 1.6mM
650 µL C1350 µL water
650 µL C1350 µL of 16 mM Congo
650 µL C1250 µL 16
mM Congo100 µL water
650 µL C1200 µL 16
mM Congo150 µL water
650 µL C1150 µL 16
mM Congo200 µL water
650 µL C1100 µL ThT
16 mM Congo
250 µL water
0.8 mM 0.4 mM 0.04 mM 0.004 mM 0.0004 mM650 µL C1
50 µL 16 mM Congo
300 µL water
650 µL C1100 µL 4 mM
Congo250 µL water
650 µL C1100 µL 0.4 mM Congo
250 µL water
650 µL C110 µL 0.4
mM Congo340 µL water
650 µL C11 µL 0.04
mM Congo349 µL water
650 µL C1250 µL 16 mM Congo100 µL water
650 µL C1250 µL 16 mM Congo100 µL water
Reduced peptide
concentration to 5mg/mL
Peptide #1
Peptide #2
Congo Red Density?
Congo Red
Recap
• Mutations in Alzheimer’s cause early onset disease
• Fluorescent dyes can be used to study amyloid interactions
• No ThT density • Unknown Congo Red density
Future Directions
• Continue to solve Congo Red interaction• Relook/ re-optimize other mutations such as:– Osaka– Iowa– Dutch
Acknowledgements• Kevin Chen• Dr. James Nowick• UROP• The Nowick Lab
• Coffee