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 molecules (enzyme/substrate)
• Motifs such as SH2, SH3, RING, coiled coil
• regulatory sequence with unknown transcription
  factor
• transcription factor with unknown target gene

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Types of Interactions

• Protein/protein
• extracellular
• intracellular
• Protein/nucleic acid

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Interaction Methods

• co-immunoprecipitation
• glutathione-S-transferase (GST) pull down
• co-purification
• chromatography, tandem affinity purification
  (TAP)
• yeast two hybrid
• phage display/expression libraries
• FRET
• solution binding- Scatchard analysis

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Co-Immunoprecipitation
B
A
IP protein A
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Tandem Affinity Purification (TAP)
Advantages - Specificity - good for complex -
PTM/localization
Drawbacks -need verification -not
quantitative -not as sensitive as 2 hyb (for
transient)
SILAC (Stable Isotope Labeling of Amino-Acid in
Cell Culture)
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Yeast Two Hybrid
DNA binding domain hybrid
Advantages -sensitivity
Activation domain encoded by a library
Drawbacks -lack of specificity -False
positives -problems with PTM -problems with
localization
Interaction
Gal1-lacZ (blue colonies)
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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Fluorescence Resonance Energy Transfer FRET
FLIM (Fluorescence lifetime imaging)
BiFC (Bimolecular fluorescence complementation)
10-50 Å, emission 1/d6
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Interaction Methods Protein/DNA

• Electrophoretic mobility shift assay (EMSA)
• SELEX
• yeast one hybrid
• Chromatin immunoprecipitation (ChIP)
• Footprinting (in vitro and in vivo)

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Electrophoretic mobility shirt assay (EMSA)
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SELEX
Random oligonucleotide pool
Affinity matrix
elute
clone sequence
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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Yeast One Hybrid
Library protein
TATA
Repoter (his, lacZ)
Bait DNA sequence
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Chromatin Immunoprecipitation (ChIP)
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Methods to Identify Gene Targets of a
Transcription Factor?

• expression profiling combined with genomic
  sequence analysis
• ChIP followed by UHTS
• SELEX combined with sequence analysis
• genetics combined with other methods

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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

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Equilibrium constant measures the 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
AB
AB/B
AB
B
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Range of Biological Dissociation Constants

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

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Phage Display