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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An experimental strategy for identifying new molecular actors in a process

  • candidate approach

  • general screen


Some situations in which this strategy could be applied actors in a process

  • 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 actors in a process

Protein/protein

  • extracellular

  • intracellular

    Protein/nucleic acid


Interaction methods
Interaction Methods actors in a process

  • 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 actors in a process

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


Tandem Affinity Purification (TAP) actors in a process

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 actors in a process

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


Fluorescence Resonance Energy Transfer: FRET actors in a process

FLIM

(Fluorescence lifetime imaging)

BiFC

(Bimolecular fluorescence complementation)

: 10-50 Å, emission ~ 1/d6


Interaction methods protein dna
Interaction Methods Protein/DNA actors in a process

  • Electrophoretic mobility shift assay (EMSA)

  • SELEX

  • yeast one hybrid

  • Chromatin immunoprecipitation (ChIP)

  • Footprinting (in vitro and in vivo)



Selex
SELEX actors in a process

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

Y actors in a process 1-n

Yeast One Hybrid

Library protein

TATA

Repoter (his, lacZ)

Bait DNA sequence



Methods to Identify Gene Targets of a Transcription Factor? actors in a process

  • 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 actors in a process

  • 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 actors in a process

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 actors in a process

  • 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


Phage display
Phage Display actors in a process


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