Discovering Macromolecular Interactions

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Discovering Macromolecular Interactions
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An experimental strategy for identifying new
molecular actors in a process

• candidate approach
• general screen

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Some situations in which this strategy could be
applied

• receptors or ligands without partners
• intracellular signaling molecules
• regulatory sequence with unknown transcription
  factor
• transcription factor with unknown target
• search for a partner of a transcription factor
• telltale motif such as SH3, RING, coiled coil

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Some Types of Interactions

• protein/protein
• extracellular
• disulphide bonds and glycosylation
• intracellular
• protein/nucleic acid

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Interaction Methods

• co-immunoprecipitation
• glutathione-S-transferase (GST) pull down
• surface plasmon resonance (BiaCore)
• FRET 10-50 Å, emmission 1/d6
• chemical cross-linking
• yeast two hybrid
• co-purification
• chromatography, centrifugation, tandem affinity
  purification (TAP)
• solution binding- Scatchard analysis

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Interaction Methods continued

• gel overlay
• phage display/expression libraries
• Electrophoretic mobility shift assay (EMSA)
• SELEX
• yeast one hybrid
• Chromatin immunoprecipitation (ChIP)
• footprinting
• filter binding

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Yeast Two Hybrid
Controls?
CHIEN, CT, BARTEL, PL, STERNGLANZ, R, AND FlELDS,
S The two-hybrid system A method to identify and
clone genes for proteins that interact with a
protein of interest. Proc. Natl. Acad. Sci. USA
Vol. 88, pp. 9578-9582, November 1991
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Tandem Affinity Purification (TAP)
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Yeast One Hybrid
Y1-n
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Methods to Identify Gene Targets of a
Transcription Factor?

• expression profiling combined with genomic
  sequence analysis
• ChIP on a chip
• SELEX combined with sequence analysis
• genetics combined with other methods

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SELEX
C.Tuerk, L. Gold Systematic evolution of
high-affinity RNA ligands of bacteriophage T4 DNA
polymerase in vitro. Science 249505-510 (1990).
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Chromatin Immunoprecipitation (ChIP)
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ChIP on a chip afterR. Young et al.
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Verifying a Putative Interaction

• demonstrate by multiple independent molecular
  methods
• co-localization
• biochemical affinity/specificity
• genetics
• phenotypic overlap between two mutants
• what is the best genetic evidence of direct
  interaction?

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Equilibrium Constant Measures Strength of
Interaction
A B
AB
AB
A B
dissociation rate koff AB
association rate kon A B
At equilibrium association rate dissociation
rate kon A B koff AB
A B koff ______ ___ KD
dissociation constant (M) AB kon
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Range of Biological Dissociation Constants
QUAL ALERT!

• adrenocorticoid receptor 10-10
• neuropeptide 10-9
• trypsin 8 x 10-5
• Ab/Ag interaction 10-5 - 10-12
• Lambda rep (monomer/dimer) 2 x 10-8
• lambda rep (dimer/DNA) 1 x 10-10

Concentration of one molecule per typical
mammalian cell volume is about 10-12 M.
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Other ways of achieving specificity of
interaction

• avidity (multivalent interaction)
• subcellular compartmentalization
• restricted expression
• activation or cofactors

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