1. Protein Complex Discovery
Who: identity of proteins in complex?
What: biological process involved?
Where: is the complex localized?
When: are proteins involved in the complex?
How much: stoichiometry of proteins in complex quantity- relative vs absolute
Regulation: modifications

2. Direct Analysis of a Complex: The Kinetochore
The kinetochore is the structure on chromosomes where the spindle fibers attach during cell division to pull the chromosomes apart
Microtubules are green, chromosomes are blue, and kinetochores pink

3. Tandem Affinity Purification
5’ gene
3’ Tag
Clone gene and add tag code to N or C terminal
Express tagged protein in cell
Bait Protein
Calmodulin
TEV cleavage site
Protein A

4. Tandem Affinity Purification (i)
The cell extract is passed over an IgG column. This binds the ProteinA tag on the protein.
The column is then washed.

5. Tandem Affinity Purification (ii)
TEV protease is added to cleave the ProtA tag away. The target is eluted and bound to a phenylsepharose column since the CaM tag binds this in the presence of calcium. The column is washed and the tagged protein released by removing calcium with EDTA

6. Cell-Map Proteomics –Tandem Affinity Purification

7. Systematic Complex pull-downs

8. Final verified complex

9. Zooming the Picture Out

10. The Big Picture

11. Yeast two-hybrid method
Goal: Determine how proteins interact with each other
Method
Use yeast transcription factors
Gene expression requires the following:
A DNA-binding domain
An activation domain
A basic transcription apparatus
Attach protein1 to DNA-binding domain (bait)
Attach protein2 to activation domain (prey)
Reporter gene expressed only if protein1 and protein2 interact with each other
The yeast two-hybrid method exploits the gene transcription machinery of yeast to study interactions between proteins in vivo. Gene transcription in S. cerevisiae, as in most eukaryotic cells, requires a DNA-binding domain, an activation domain, and the basic transcription apparatus. A protein of interest (call it protein1) is attached to the DNA-binding domain of a well-characterized transcription factor such as Gal4. This protein is called the “bait.” Protein2 is attached to the activation domain from the same transcription factor. The protein2-activation domain complex is called the “prey.”If protein1 and protein2 interact with each other, then DNA binding and activation will occur and a reporter gene will be expressed. The yeast two-hybrid method is shown schematically in the next slide.

12. A schematic of the yeast two-hybrid method
The figure in the slide shows in greater detail how the yeast two-hybrid method works. Two sets of yeast colonies are grown. In this example, the first set of colonies consists of all yeast open reading frames (ORFs) attached to the DNA-binding domain. The second set of colonies consists of all ORFs attached to the activation domain. Yeast from the two sets of colonies are mated with each other to produce every possible protein–protein interaction in the offspring. Cells expressing the reporter gene are selected, and their ORFs are identified by DNA sequencing to reveal the precise protein–protein interaction. This method has been successfully applied to determine all protein–protein interactions in the yeast Saccharomyces cerevisiae, as shown in the next slide.

13. Drosophila interaction map
Recently a group of researchers from CuraGen Corporation, a biotechnology company, completed an extensive protein-interaction map of Drosophila, using the yeast two-hybrid system. The image in this slide shows the entire interaction map as well as close-up of a particular region. The nodes of the interaction map are coded to indicate the likely function of the protein they represent. Black stars around a node indicate a gene implicated in human disease. For this map, the 3,000 interactions with the highest confidence values were chosen, and they link 3,522 proteins.

14. Attaching a Green Flurescent Protein to an ORF
PCR product
GFP
HIS3MX6
Homologous
recombination
Chromosome
ORF1
ORF2
As in the case of protein–protein interactions, the yeast genome was used to determine the subcellular location of products of all ORFs. This was done by inserting the sequence for the green fluorescent protein (GFP) together with a marker gene (HIS3MX6) that makes it possible to select transformed yeast strains in histidine-free medium. Oligonucleotide primers corresponding to each ORF were used to generate ORF-specific sequences flanking the GFP and selectable marker. This approach permits the sequence to be incorporated into the chromosomal DNA via homologous recombination. The fusion protein resulting from this insertion has the GFP attached at the C-terminus of the ORF protein. Inside the living cell, the precise location of the protein can be determined by localizing the protein’s fluorescence.
Fusion protein
NH2
protein
GFP
COOH

15. Location of proteins revealed
75% of yeast proteome localized
> 40% of proteins in cytoplasm
67% of proteins were previously unlocalized
Localizations correlate with transcriptional modules
cytoplasm
nucleus
The image in this slide shows a tagged protein that was localized to the nucleus. Note the contrast between the glowing nucleus and the much fainter cytoplasm. This study was able to successfully localize 75% of the entire yeast proteome, with greater than 40% of the proteins being found in the cytoplasm, and other proteins being localized in 21 distinct subcellular regions, including the nucleus, mitochondria, and the endoplasmic reticulum. The researchers were also able to show that subcellular localizations frequently correlated with transcriptional modules. By comparing their data with previous work on protein–protein interactions, they were also able to show that proteins localized in different regions had different probabilities of interacting with each other. For example, proteins in the cytoplasm were only 1.3 times more likely to interact with each other by chance, while microtubule-bound proteins were 56 times more likely to do so.
A protein localized
to the nucleus

16. Subcellular localization of interacting proteins
The Drosophila protein-interaction map was also organized to show where interactions are likely to occur in the cell. Proteins with known, or annotated, subcellular localizations were used to determine the sites of likely interactions. The nodes are color coded to indicate what cellular component each protein belongs to. In this map, there are 2,346 proteins and 2,268 interactions.

17. Molecular Machinary-Parts List