Photoreactive Crosslinker Chemistry
Photo-activatable (or photo-chemical) crosslinking reactions require energy from light to initiate. Photoreactive groups are chemically inert compounds that become reactive when exposed to ultraviolet or visible light. Practically all varieties of photoreactive groups used in reagents for crosslinking applications require exposure to ultraviolet light (UV light) for molecular activation.
Photochemical reactive groups have certain advantages over strictly thermochemical reagents for crosslinking and labeling applications with biological samples and experiments. Most importantly, they make it possible to add reagent at an early step in an experiment and then to initiate crosslinking (by exposure to UV light) at some later step that coordinates with the particular biological condition of interest. Additionally, many of these groups will conjugate to any one of several common functional groups in proteins that they encounter during the brief time when they are activated. This feature makes them particularly useful in capturing protein interactions (see discussion below).
Photoreactive groups that have been incorporated into crosslinking and labeling compound for use in bioconjugate techniques include aryl azides, azido-methyl-coumarins, benzophenones, anthraquinones, certain diazo compounds, diazirines, and psoralen derivatives. The most useful of these for protein biology research are aryl azides and diazirines.
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When an aryl azide is exposed to UV light (250 to 350nm), it forms a nitrene group that can initiate addition reactions with double bonds, insertion into C–H and N–H sites, or subsequent ring expansion to react with a nucleophile (e.g., primary amines). The latter reaction path dominates when primary amines are present in the sample.
Thiol-containing reducing agents (e.g., DTT or 2-mercaptoethanol) must be avoided in the sample solution during all steps before and during photo-activation, because they reduce the azide functional group to an amine, preventing photo-activation. Reactions can be performed in a variety of amine-free buffer conditions. If working with heterobifunctional photoreactive crosslinkers, use buffers compatible with both reactive chemistries involved. Experiments must be performed in subdued light and/or with reaction vessels covered in foil until photoreaction is intended. Typically, photo-activation is accomplished with a hand-held UV lamp positioned close to the reaction solution and shining directly on it (i.e., not through glass or polypropylene) for several minutes.
Three basic forms of aryl azides exist: simple phenyl azides, hydroxyphenyl azides, and nitrophenyl azides. Generally, short-wavelength UV light (e.g., 254nm; 265 to 275nm) is needed to efficiently activate simple phenyl azides, while long-UV light (e.g, 365nm; 300 to 460nm) is sufficient for nitrophenyl azides. Because short-wave UV light can be damaging to other molecules, nitrophenyl azides are usually preferable for crosslinking experiments.
Although homobifunctional aryl azide crosslinkers were commercially available in the past, they have limited utility compared to alternatives and are no longer sold (search literature for "BASED, bis-[β-(azidosalicylamido)ethyl]disulfide)"). Nearly all applications for aryl azide reagents involve heterobifunctional chemistries in which an aryl azide group is paired opposite a different type of reactive group, such as an amine-reactive NHS ester. These compounds are used for a variety of bait-and-prey strategies to investigate protein-protein interactions or protein-nucleic acid interactions.
1. Capture protein interactions
Heterobifunctional NHS-ester/aryl-azide crosslinkers are used in experiments to discover or analyze the conditions in which a particular protein interaction occurs.
Suppose a researcher has a purified protein (X) and wishes to compare two conditions for relative abundance of a second protein (Y), which the researcher knows is the direct binding partner of X. First, the crosslinker is reacted in isolation (and in subdued light) with X; the crosslinker attachs at its NHS-ester end to surface primary amines of X, labeling X with several ready-to-activate aryl azide groups. After desalting X to remove non-reacted crosslinker, X is added to samples of Y that represent different treatment conditions (e.g., cell lysates prepared from cells grown in different conditions). Finally, once sufficient time has passed for X and Y to bind one another, the sample is irradiated with UV light to activate the aryl azide moiety, which then conjugates to any protein functional group it is near.
Where nearby amino acids are those of the binding partner (Y), covalent crosslinks between X and Y will form. At this point, the results can be analyzed in several ways. Assuming that the researcher has a specific antibody to detect X, the products can be analyzed by electrophoresis and Western blotting. Conjugated proteins will run as one larger protein rather than separate individual proteins, and this difference could be detected and quantified.
Depending on the spacer length and cleavability features of the crosslinker, different particular pairs of protein interactors can be more or less effectively conjugated and analyzed in different ways. Aryl azide compounds that are heterobifunctional with amine-reactive, sulfhydryl-reactive and carbonyl-reactive groups are commercially available.
2. Label protein interactions
Label-transfer is an extension of the heterobifunctional crosslinking just described and is used to investigate protein interactions. Besides the two crosslinking ends, these reagents incorporate a detectable tag or label (e.g., a fluorophore or biotin) and a cleavable spacer arm (usually a disulfide bond).
Once pairs of interacting proteins have been crosslinked (as described above for hypothetical proteins X and Y), the spacer arm connecting them can be cleaved. This separates the proteins but leaves the label (biotin in the case of Sulfo-SBED) attached to Y. Thus, the biotin label was effectively transferred from the "bait" protein (X) to the "prey" protein (Y).
Diazirines are a newer class of photo-activatable chemical groups that are being incorporated into various kinds of crosslinking and labeling reagents. The diazirine (azipentanoate) moiety has better photostability than phenyl azide groups, and it is more easily and efficiently activated with long-wave UV light (330 to 370nm).
Photo-activation of diazirine creates reactive carbene intermediates. Such intermediates can form covalent bonds through addition reactions with any amino acid side chain or peptide backbone at distances corresponding to the spacer arm lengths of the particular reagent.
Although aryl azide reagents are more widely cited in the literature, this is likely to change as diazirine reagents become more widely available and gradually replace aryl azide in most applications.
1. Capture protein interactions
Where diazirine equivalents of heterobifuncational aryl azide reagents are available, all of the same kinds of protein interaction experiments are possible. Currently, several varieties diazirine compounds are available that have an amine-reactive NHS ester at the opposite end.
2. Metabolic labeling and crosslinking
The stability and very small size of the diazirine group also enable crosslinking experiments that involve metabolic labeling. For example, Photo-L-Leucine and Photo-L-Methionine are analogs of native amino acids that contain the diazirine group in their side chains. When these compounds are added to culture media instead of their native counterparts, protein synthesis machinery will use the photoreactive versions to build proteins. In this way, proteins themselves become the crosslinking reagents for in vivo crosslinking strategies.
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