Download Unbinding forces of single antibody-antigen

Unbinding forces of single antibodyantigen complexes correlate with
their thermal dissociation rates
Schwesinger et al, 2000
Jen Chao
20.309 Presentation
November 20, 2008
Outline
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Background
Methods
Results
Further Research
Motivation
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Correlation between unbinding force and
solution kinetics or thermodynamics
Prediction of off-rates
Combinatorial chemistry, genomics research
Background
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Atomic Force Microscopy (AFM)
Antibodies specific for fluorescein
Parameters: KD = koff/kon
Methods
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3 fluorescein-binding antibodies, point
mutations
KD, kon, koff for each protein in solution
Dependence on Pulling velocity
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Nonlinearity
Off/On-Rate Measurements
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Competitive dissociation assay
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1nM fluorescein solution
Analog mixed in
Stopped-flow fluorimeter
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5 different concentrations of scFv
Activation E, Loading Rates
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EA ~ off-rate temperature dependence
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10, 15, 20, 25 degrees C
Force-distance measurements
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Constant loading rate
Loading rate dependent
Results
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Remarkable correlation of the unbinding
forces to the off-rates.
Off vs. On Rate
Off-rate follows Arrhenius Law:
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V and EA are correlated
Further research
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Some protein mutants break earlier along
unbinding path
Recommend How mutants affect interaction
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Geometry
Protein properties
Conclusion: Correlation between off-rate and
unbinding force