Prey protein

Sample containing prey protein inleraclion partner. 

Another crosslinker, SAED (Chapter 5, Section 3.9), can be used in a similar fashion, but instead of transferring a radioactive label, it contains a fluorescent portion that is transferred to a binding molecule after cleavage. Similarly, sulfo-SBED routinely is used to study protein interaction. Cleavage of a disulfide bridge after capture of interacting proteins results in transfer of a biotin label to the unknown prey protein (Chapter 28, Section 3.1). The biotin modification then can be used to detect or isolate the unknown interactor for subsequent identification. 

Add the modified bait protein to a sample containing potentially interacting prey proteins and incubate for 1 hour protected from light. The sample may be cells (for cell-surface interaction studies), cell lysate, or various extracts from cells, tissues, or biological fluids. 

Figure 28.11 Sulfo-SBED is a label transfer agent that contains a water-soluble sulfo-NHS ester to label bait proteins and a phenyl azide group for photoreactive capture of a prey protein. The biotin label can be used for detection or isolation of protein-protein conjugates using (strept)avidin reagents. The stars indicate the atoms that were used to measure the indicated molecular dimensions.

Figure 28.12 Sulfo-SBED first is used to label a bait protein through reaction of the sulfo-NHS ester with available amine groups on the protein, yielding an amide bond linkage. This labeled bait protein then is added to a sample containing proteins that potentially could interact with the bait. After an incubation period, the sample is exposed to UV light to photoactivate the phenyl azide group. This reaction causes any interacting prey proteins to be crosslinked with the bait protein, forming a complex containing a biotin affinity tag.

Biotin label transferred to unknown prey protein... 

Figure 28.14 A trifunctional label transfer reagent containing a thiol-reactive pyridyl disulfide group, a photo-reactive phenyl azide, and a biotin affinity tag. This compound can be used to label bait proteins through available thiol groups and capture interacting prey proteins by photoreactive conjugation.

Add the labeled bait protein to a sample containing the putative interacting prey proteins. The quantity of bait protein to be added to a given sample should be within the same concentration level as the amount of prey proteins present. The optimal level of addition may have to be determined by varying the amount of bait protein concentrations in a number of sample solutions to decide which concentration results in the best interaction complexes being formed. 

Figure 28.15 Two similar label transfer reagents containing a thiol-reactive methanethiolsulfonate group to label bait protein through available sulfhydryls, a tetrafluorophenyl azide group for high-efficiency photoreac-tive conjugation with interacting prey proteins, and a long biotin affinity tag.

Prey protein cleaved near FeBABE modification sites on bait protein... 

In order to facilitate analysis of FeBABE produced fragments, the prey protein or biomolecule is labeled at one end with a tag that can be detected after electrophoresis, usually in a transfer blot. The tag can be a fusion tag, such as 6X His, or any other group that can be targeted with an antibody and detected. Alternatively, radiolabels and fluorescent labels have been used with prey molecules, including the use of end-labeled DNA to study where DNA binding proteins dock onto the oligonucleotide sequence. 

The fragments formed by FeBABE fragmentation are analyzed by comparing them to enzymatic or chemical cleavage patterns observed by treatment on the same prey protein. Since the cleaved prey protein is detected by its end-labeled tag, the only fragments detected are those... 

Dissolve or buffer exchange the prey protein into 50mM MOPS, 120mM NaCl, 0.1 mM EDTA, 10mM MgCl2, 10 percent glycerol, pH 8.0. 

Mix the labeled bait protein with the prey protein at equal molar ratios and incubate for 30 minutes at room temperature to create protein interaction complexes. 

Analyze the cleaved proteins by SDS gel electrophoresis and Western blotting, followed by specific detection of the prey protein label. Comparison of prey protein fragments formed by chemical and enzymatic digestion can yield information as to the site of the resultant FeBABE-mediated cleavage. 

As mentioned in Chapter 2 for the description of our Y2H library screening procedure, what we obtained after a bioinformatics analysis is the location of the interacting domain within the bait or the prey protein. In reality, we do not measure interaction between full length proteins but between domains within proteins which is a closer representation of an in vivo interaction where only some key fragments of each protein interact with one another in space. These domains were called Selected Interacting Domain or SID. It is therefore clear that bioinformatics sequence analysis that compare SIDs obtained from Y2H experiments with Interpro domains can lead to very interesting observation regarding the domain keen to interact and can help to discriminate more specifically true and false positives. 

Figure 4 Architecture of three-hybrid screens, (a) The classic, small-molecule-based, three-hybrid platform is shown. The three components of this systems are a DNA-binding protein fused to a known drug-binding domain (X), a bifunctional small molecule that binds X on one end and displays a query "bait" on the other, and a library of possible "prey" proteins (V) expressed as fusions to transcriptional activation domains. Assembly of the ternary complex recruits the activation domain to the promoter and initiates reporter gene expression. A partial list of examples is shown in the inset boxes because of space constraints, some relevant systems are absent, (b) The RNA-based, three-hybrid is assembled in a similar fashion except that the bifunctional ligand is an RNA molecule. The complex between the MS2 RNA and the MS2-coat protein is typically used as the anchoring interaction.

---