LOCALIZATION OF A MOLECULE - Stanford University

LOCALIZATION OF A MOLECULE - Stanford University

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)

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 WCE IP A A

Control IP WCE IP A B IP protein A

Resolve Immune Complex by SDS PAGE WesternBlot with Antibody against B Tandem Affinity

Purification (TAP) dvantages Specificity good for complex PTM/localization acks

verification quantitative as sensitive as 2 hyb (for transient) SILAC (Stable Isotope Labeling of Amino-Acid in Cell Cult Yeast Two Hybrid

DNA binding domain hybrid Advantages -sensitivity Activation domain encoded by a library awbacks

ack of specificity alse positives roblems with PTM roblems 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 imagin 10-50 , emission ~ 1/d6 BiFC (Bimolecular fluorescence complementation)

Interaction Methods Protein/DNA Electrophoretic mobility shift assay (EMSA) SELEX yeast one hybrid Chromatin immunoprecipitation (ChIP)

Footprinting (in vitro and in vivo) Electrophoretic mobility shirt assay (EMSA) SELEX Random oligonucleotide pool

Affinity matrix elute clone & sequence .Tuerk, L. Gold Systematic evolution of high-affinity RNA ligands of

acteriophage T4 DNA polymerase in vitro. Science 249:505-510 (1990). Yeast One Hybrid Y1-n Library protein TATA Bait DNA sequence

Repoter (his, lacZ) Chromatin Immunoprecipitation (ChIP) 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

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 association rate =

At equilibrium: AB AB A + B kon [A] [B] dissociation rate = koff [AB]

association rate kon [A] [B] dissociation rate koff [AB] [AB]/[B]

koff ___ = KD = dissociation constant (M) kon [AB] [A] [B] ______ =

[AB] = = [B]

[AB] 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 Phage Display

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