Label Transfer Chemistry for the Characterization of Protein−Protein Interactions

Abstract

A new label transfer method is presented that overcomes most of the limitations of current systems. A protein of interest is tagged with tetra-cysteine sequence (FlAsH Receptor Peptide (FRP)) that binds tightly and specifically to a chimeric molecule 3,4-dihydroxyphenylalanine-biotin-4′,5′-bis (1,3,2-dithioarsolan-2-yl)fluorescein (DOPA-biotin-FlAsH). Upon brief periodate oxidation, the DOPA moiety is cross-linked to nearby surface-exposed nucleophiles. Boiling the products in excess dithiol dissolves the FlAsH-FRP interaction, resulting in transfer of the biotin tag to the partner proteins, allowing them to be identified by standard methods. Most methods to characterize protein-protein interactions require the protein of interest to be displayed as an artificial fusion outside of its native environment. To overcome such limitations, a biochemical method called label transfer has been developed, in which a latent cross-linking agent and some easily detectable tag such as a biotin or a radiolabel are connected to the protein of interest via a cleavable linker. Once the modified protein is incorporated into its native complex, the cross-linking moiety is activated and the linker arm is then cleaved, resulting in transfer of the tag to the partner protein 1. While this approach has been employed in the study of some protein complexes, 2,3 the utility of current label transfer technology is severely limited by the requirement to modify the protein of interest covalently and then reconstitute the complex of interest with the modified protein. The inefficient cross-linking chemistry commonly used in such systems (usually photolysis of aryl azides) also complicates the characterization of partner proteins 3. We report here a new type of label transfer system that eliminates the need for covalent modification of the protein of interest (see Scheme 1). It involves tagging the "bait" protein with a tetracysteine-containing peptide (CCPGCC, here called FRP for FlAsH Receptor

Physical and Functional Interactions of Monoubiquitylated Transactivators with the Proteasome

“…␣-Rpn1 and ␣-Rpn2 were produced in mouse. The DOPA-biotin-FlAsH chimera (DBF) cross-linking reagent (19) and the DOPA-Gal4 AD peptide have been described (20).…”

“…The proteins indicated in the figure were added to the reaction mix at the same time as peptide addition. Cross-linking to detect mono-Ub/ proteasome interaction was done as described (19) with the following changes. 110 nM 26 S or 19 S proteasome was mixed with 10 M CCPGCC-Ub in TR reaction buffer.…”

“…Ub Binds Directly to the 19 S RP-To distinguish whether the activator or the proteasome is the target of free Ub, we turned to a novel type of cross-linking/label transfer chemistry developed recently in our laboratory (19). In this method ( Fig.…”

Collision-induced dissociation of lys-lys intramolecular crosslinked peptides

“…Because both fragmentation spectra were identical, it can be concluded that this species is the intermolecular crosslinked peptide. Figure 3 presents MALDI-MS/MS annotated spectra of intramolecular crosslinked P1c, P2c, and P3c (for noScheme 3 menclature see Liu et al [38]). These peptides can be seen as branched cyclic peptides because the DSSpeptide bond is also an amide bond.…”

How to obtain labeled proteins and what to do with them

“…A particularly pressing problem in biology is the study of protein interactions. A previously introduced approach based on the tetracysteine tag ( Figure 3a) uses a trifunctional compound containing a biarsenical dye to bind to the protein of interest, a crosslinker triggered by addition of sodium periodate to induce tethering to binding partners, and biotin for detection of the interaction partner after SDS-PAGE and western blotting [52]. This affinity labeling method has recently been applied to study the interaction between ubiquitin and the proteasome [53].…”