discovering macromolecular interactions
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Discovering Macromolecular Interactions. An experimental strategy for identifying new molecular actors in a process. candidate approach general screen. Some situations in which this strategy could be applied. receptors or ligands without partners intracellular molecules (enzyme/substrate)

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Presentation Transcript
slide2

An experimental strategy for identifying new molecular actors in a process

  • candidate approach
  • general screen
slide3

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
types of interactions
Types of Interactions

Protein/protein

  • extracellular
  • intracellular

Protein/nucleic acid

interaction methods
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
co immunoprecipitation
Co-Immunoprecipitation

Control

IP

IP

A

WCE

Control

IP

IP

A

WCE

B

A

IP protein A

Western-

Blot with

Antibody against B

Resolve Immune

Complex by SDS PAGE

slide7

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)

yeast two hybrid
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

slide9

Fluorescence Resonance Energy Transfer: FRET

FLIM

(Fluorescence lifetime imaging)

BiFC

(Bimolecular fluorescence complementation)

: 10-50 Å, emission ~ 1/d6

interaction methods protein dna
Interaction Methods Protein/DNA
  • Electrophoretic mobility shift assay (EMSA)
  • SELEX
  • yeast one hybrid
  • Chromatin immunoprecipitation (ChIP)
  • Footprinting (in vitro and in vivo)
selex
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 249:505-510 (1990).

yeast one hybrid

Y1-n

Yeast One Hybrid

Library protein

TATA

Repoter (his, lacZ)

Bait DNA sequence

slide15

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
verifying a putative interaction
Verifying a Putative Interaction
  • Demonstrate by multiple independent molecular methods
    • co-localization
    • biochemical affinity/specificity
  • Genetics
    • phenotypic overlap between two mutants
equilibrium constant measures the strength of interaction
Equilibrium constant measures the strength of interaction

A + B

AB

AB

A + B

association rate = kon [A] [B]

dissociation rate = koff [AB]

At equilibrium: association rate = dissociation rate

kon [A] [B] = koff [AB]

[A] [B] koff

______= ___ = KD = dissociation constant (M)

[AB] kon

[AB]/[B]

[AB]

[AB]

[B]

range of biological dissociation constants
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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