1. (D) Crosslinking Interacting proteins can be identified by crosslinking. A labeled crosslinker is added to protein X in vitro and the cell lysate is added so that interactions can occur. If the crosslink is activated at this stage, interacting proteins become covalently attached to the bait. After purification, the crosslink can be cleaved and the interacting proteins separated by 2D SDS-PAGE.

2. Crosslinking (contd.)

3. Physical methods High-resolution methods: (e.g., X-ray crystallography & NMR) providing data about the relative spacing of atoms of interacting molecules. Low-resolution methods: e.g., electron crystallography and electron tomography.

4. FRET (Fluorescence Resonance Energy Transfer)

5. FRET FRET is the energy transfer that occurs when two fluorophores are close together, and one of fluorophores (the donor) has an emission spectrum that overlaps the excitation spectrum (absorption spectrum) of the other fluorophoe (the acceptor).

6. Basic Theory of FRET: k T (r) = (Q D  2 )(1/  D r 6 )(9000 *In10)(1/128  5 N A n 4 )( ∫ F D ( )  A ( ) 4 d ) = (1/  D )(R 0 /r) 6 where R 0 is the Förster distance r is the distance between the donor and the acceptor E = 1/(1+(r/R 0 ) 6 ) where E is the efficiency of the energy transfer J(), the so-called overlap integral= ∫F D () A () 4 d

7. FRET

8. FRET R 0 : is the F ö rster distance

9. FRET: distance- dependent Note: when r=R 0, E=0.5 r is the distance between the donor and the acceptor R 0 is the F ö rster distance E is the efficiency of the energy transfer F D : the fluorescence intensity of the donor in the absence of the acceptor F DA : the fluorescence intensity of the donor in the presence of the acceptor

10. Library-based methods for the global analysis of binary interactions Standard cDNA expression libraries Phage display method The yeast two-hybrid system

11. Standard cDNA expression libraries Expression libraries are usually screened with labeled antibodies. In place of antibodies, other proteins can be used as probes. For example, labeled calmodulin has been used to screen for calmodulin-binding proteins. Low throughput Does not provide the native conditions for the folding of all proteins, so a significant number of interactions would not be detected.

12. Phage display method (1) M13 (a filamentous phage containing ss-DNA encased in a protein coat): contains five coat proteins, two of which are gVIIIp (gene VIII protein) and gIIIp (gene III protein).

13. Phage display method (2)

14. Phage display method (2): contd.

15. The phage display method

16. The yeast two-hybrid system Transcription factors generally comprise two functionally independent domains, one for DNA binding and one for transcriptional activation. These do not have to be covalently joined together, but can be assembled to form a dimeric protein. This principle is exploited to identify protein interactions. Bait proteins are expressed in one yeast strain as a fusion with a DNA-binding domain and candidate prey proteins are expressed in another strain as fusions with a transactivation domain. When the two strains are mated, functional transcription factors are assembled only if the bait and prey interact. This can be detected by including a reporter gene activated by the hybrid transcription factor.

17. The yeast two-hybrid yeast

18. Limitations of the yeast two-hybrid system First, where independent groups have carried out similar, large-scale studies, the degree of overlap in the reported interactions is very low (10- 15%). This suggest either that the screens were not comprehensive or that even minor differences in experimental conditions could influence the types of interactions that are detected.

19. Limitations of the yeast two-hybrid system Secondly, a significant number of well-characterized interactions are not detected in the large-scale screens, suggesting there is a high level of false negatives. Thirdly, a significant number of interactions that are detected in large-scale screens appear spurious when investigated in more detail, suggesting there is also high level of false positives.

20. A variant of the yeast two-hybrid system

21. Node: proteins or protein complexes are treated as nodes. Edge (or link): interactions between them. Some proteins serve as hubs for very large numbers of interactions. Protein interaction maps

22. Binary interaction map including 1200 interacting proteins in yeast Trends in Cell biology (2001), 11: 102-106

23. A simplified version in which yeast proteins have been clustered according to their function