(D) Crosslinking(D) CrosslinkingInteracting 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.

Crosslinking (contd.)Crosslinking (contd.)

Physical methodsPhysical methodsHigh-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.

FRET (FRET (FFluorescence luorescence RResonance esonance EEnergy nergy TTransfer)ransfer)

FRETFRETFRET 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).

Basic Theory of FRET:kT(r) = (QD2)(1/Dr6)(9000 *In10)(1/1285NAn4)(∫FD()A() 4d ) = (1/D)(R0/r)6 where R0 is the Förster distance r is the distance between the donor and the acceptor

E = 1/(1+(r/R0)6)

where E is the efficiency of the energy transfer

J(), the so-called overlap integral= ∫FD()A() 4d

FRETFRET

FRETFRET

R0: is the Förster distance

FRET: distance-FRET: distance-dependentdependent

Note: when r=R0, E=0.5

r is the distance between the donor and the acceptor

R0 is the Förster distance

E is the efficiency of the energy transfer

FD: the fluorescence intensity of the donor in the absence of the acceptorFDA: the fluorescence intensity of the donor in the presence of the acceptor

Library-based methods for the Library-based methods for the global analysis of binary global analysis of binary

interactionsinteractions

Standard cDNA expression libraries

Phage display method

The yeast two-hybrid system

Standard cDNA Standard cDNA expression librariesexpression 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 throughputDoes not provide the native conditions for the folding of all proteins, so a significant number of interactions would not be detected.

Phage display method Phage display method (1)(1)

M13 (a filamentous phage containing ss-DNA encased in a proteincoat): contains five coat proteins, two of which aregVIIIp (gene VIII protein) and gIIIp (gene III protein).

Phage display method Phage display method (2)(2)

Phage display method Phage display method (2): contd.(2): contd.

The phage display method

The yeast two-hybrid The yeast two-hybrid systemsystem

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.

The yeast two-hybrid yeast

Limitations of the yeast Limitations of the yeast two-hybrid systemtwo-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.

Limitations of the yeast Limitations of the yeast two-hybrid systemtwo-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.

A variant of the yeast A variant of the yeast two-hybrid systemtwo-hybrid system

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

Binary interaction map including 1200 interacting proteins in yeast

Trends in Cell biology (2001), 11: 102-106

A simplified version in which yeast A simplified version in which yeast proteins have been clustered proteins have been clustered

according to their functionaccording to their function