Discovering Macromolecular Interactions - PowerPoint PPT Presentation

About This Presentation
Title:

Discovering Macromolecular Interactions

Description:

Random oligonucleotide pool Affinity matrix Yeast One Hybrid Y1-n Bait DNA sequence Library protein TATA Repoter (his, lacZ) Chromatin Immunoprecipitation ... – PowerPoint PPT presentation

Number of Views:117
Avg rating:3.0/5.0
Slides: 33
Provided by: Bioinf54
Learn more at: http://web.stanford.edu
Category:

less

Transcript and Presenter's Notes

Title: Discovering Macromolecular Interactions


1
Discovering Macromolecular Interactions
2
An experimental strategy for identifying new
molecular actors in a process
  • candidate approach
  • general screen

3
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

4
Types of Interactions
  • Protein/protein
  • extracellular
  • intracellular
  • Protein/nucleic acid

5
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

6
Co-Immunoprecipitation
B
A
IP protein A
7
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)
8
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
9
Fluorescence Resonance Energy Transfer FRET
FLIM (Fluorescence lifetime imaging)
BiFC (Bimolecular fluorescence complementation)
10-50 Ã…, emission 1/d6
10
Interaction Methods Protein/DNA
  • Electrophoretic mobility shift assay (EMSA)
  • SELEX
  • yeast one hybrid
  • Chromatin immunoprecipitation (ChIP)
  • Footprinting (in vitro and in vivo)

11
Electrophoretic mobility shirt assay (EMSA)
12
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).
13
Yeast One Hybrid
Library protein
TATA
Repoter (his, lacZ)
Bait DNA sequence
14
Chromatin Immunoprecipitation (ChIP)
15
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

16
Verifying a Putative Interaction
  • Demonstrate by multiple independent molecular
    methods
  • co-localization
  • biochemical affinity/specificity
  • Genetics
  • phenotypic overlap between two mutants

17
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
18
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

19
(No Transcript)
20
(No Transcript)
21
(No Transcript)
22
(No Transcript)
23
(No Transcript)
24
(No Transcript)
25
(No Transcript)
26
(No Transcript)
27
(No Transcript)
28
(No Transcript)
29
(No Transcript)
30
(No Transcript)
31
(No Transcript)
32
Phage Display
Write a Comment
User Comments (0)
About PowerShow.com