Map interaction sites between two purified and well-characterized individual proteins. Attach the FeBABE ("Iron Babe") cutting agent to sulfhydryl groups of one protein, then activate the cutting (artificial protease) function to cleave the interacting protein at the binding domain.

Co-immunoprecipitations, pull-down assays, far-Western analysis and label transfer reactions are excellent and widely used in vitro approaches to discover or confirm protein interactions. These methods verify that an interaction may exist in vivo between the binding partners identified. The FeBABE artificial protease reagent and Pierce Protein Interaction Mapping Kit enable more detailed characterization of protein interaction sites. The kit assembles the FeBABE reagent and other essential reagents and buffers to bring success to both first time and experienced users of this technology. The user provides the purified bait protein, a labeled prey protein and a prey-detecting antibody.

What is FeBABE " artificial protease"?

FeBABE (referred to as "Iron Babe") is the reagent Fe(III) (S)-1-(p-Bromoacetamido-benzyl)ethylene diamine tetraacetic acid (Figure 1). FeBABE is a sulfhydryl-reactive reagent that incorporates an Fe(III) EDTA moiety into a purified bait protein. The bromoacetyl group allows the reagent to be covalently attached to proteins at sulfhydryl groups. When activated by ascorbate and peroxide, the Fe(III) EDTA group will cleave peptide bonds, acting as an artificial protease.

Figure 1. Chemical structure of FeBABE reagent.

How does one use FeBABE to map protein interactions?

First, the FeBABE reagent is attached via its bromoacetyl group to sulfhydryl sites of a purified "bait" protein. Sulfhydryl (-SH) groups may occur naturally as native cysteine residues, or can be introduced by site-directed mutagenesis or by conversion of ε-NH₂ groups of lysine with 2-iminothiolane. This attaches the cutting function onto specific sites on the bait protein, effectively turning this first protein into a "cutting protein" or artificial protease.

Second, the bait/cutting protein is added to the known interacting protein or polypeptide. Upon establishment of the binding interaction, the Fe(III) chelate (cutting) moiety is activated, causing peptide bonds within reach (12 Å) of the labeled bait protein sites to be cleaved. In the presence of ascorbate and peroxide, the Fe³⁺ EDTA portion of the reagent is reduced to Fe²⁺. This reduction promotes the cleavage of peptide bonds by the bait protein in a non-sequence specific manner. (Figure 2). When the "prey" or "target" protein binds to the bait, peptide bonds on the prey in proximity of the bait-tethered Fe²⁺ (EDTA) are cleaved.

Finally, the resulting peptide pattern, when analyzed by electrophoresis, immunoblotting, sequencing or mass spectrometry techniques, can provide information relating to the region of contact within the interacting complex.

Requirements for the use of the FeBABE artificial protease to map protein interactions: Two purified proteins (bait and prey), preferably of known sequence, that are confirmed or putative binding partners. Bait protein is modifiable with FeBABE, enabling it to act as the cutting protein. Prey protein is end-labeled. Several label-detection options, such as radiolabeling, fluorophore labeling or expression tagging, are available to the user. Additionally, sequence-specific antibodies against the N or C terminal region of the prey will enable detection of the resulting peptides. When expression tagging is used, antibodies against the tag impart the ability to detect the resulting peptides in a Western blot analysis.
		Figure 2. Peptide bond cutting mechanism of FeBABE modified bait protein.

Mapping the interaction

To map the interaction site, the cleavage pattern is compared with the unbound end-labeled target protein and known mobility standards. Molecular weight markers can be used for an initial approximation of size. More accurate determination of the residues involved in the binding site requires comparison to proteolytic fragments of the target protein (cleavage standards) that can be created using site-specific enzymatic and/or chemical cleavage (e.g., CNBr or BNPS Skatole). Alternatively, the target protein can be engineered with a series of truncations. The region/locus at which the cut occurred is assigned for each fragment by comparing it against the cleavage standards. The outcome of this method is a map (3-D, if the tertiary structure is known) of residues in or near the site of interaction on the target protein.

Figure 3. Analysis of peptide mapping by Western blot. Western blot using antibody directed to the N-terminal epitope of the target protein. Comparison of FeBABE cleavage of the interaction site with cleavage standards and negative reaction controls. Lane 1. Chemical cleavage of target protein with CNBr at methionine residues. Lane 2. Chemical cleavage of target protein with BNPS-skatole at tryptophan residues. Lane 3. Reaction mixture containing target protein, FeBABE-cutting protein conjugate and activators (ascorbate and peroxide). Lane 4. Reaction mixture containing target protein and FeBABE-cutting protein conjugate without activators (negative control). Lane 5. Reaction mixture containing target protein, unconjugated cutting protein (no FeBABE) and activators (negative control).

Highlights

• Kit is a complete reagent set – pre-formulated, pre-measured and validated reagents provide better control over the reaction than homemade reagents and buffers
• System designed to maintain native protein structures – FeBABE bait conjugation conditions are non-denaturing; nonspecific peptide bond cleavage occurs under mild conditions
• Peptide bonds are cut rapidly and with high yield – obtain peptide fragments in sufficient quantity for gel and Western blot analysis
• Bait protein flexibility – use with bait proteins containing endogenous -SH groups, expressed with -SH groups inserted using site-directed mutagenesis, having surface amine groups that can be converted to -SH groups with modification reagents such as 2-iminothiolane.

References

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