Download
1 / 32
320 likes | 378 Views
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)
E N D
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) • Motifs such as SH2, SH3, RING, coiled coil • regulatory sequence with unknown transcription factor • transcription factor with unknown target gene
Types of Interactions Protein/protein • extracellular • intracellular Protein/nucleic acid
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 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) 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 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 FLIM (Fluorescence lifetime imaging) BiFC (Bimolecular fluorescence complementation) : 10-50 Å, emission ~ 1/d6
Interaction Methods Protein/DNA • Electrophoretic mobility shift assay (EMSA) • SELEX • yeast one hybrid • Chromatin immunoprecipitation (ChIP) • Footprinting (in vitro and in vivo)
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).
Y1-n Yeast One Hybrid Library protein TATA Repoter (his, lacZ) Bait DNA sequence
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 • Demonstrate by multiple independent molecular methods • co-localization • biochemical affinity/specificity • Genetics • phenotypic overlap between two mutants
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 • 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
