Protein interactions crosslinkers

The modification of amino acids in proteins and peptides by oxidative processes plays a major role in the development of disease and in aging (Halliwell and Gutteridge, 1989, 1990 Kim et al., 1985 Tabor and Richardson, 1987 Stadtman, 1992). Tissue damage through free radical oxidation is known to cause various cancers, neurological degenerative conditions, pulmonary problems, inflammation, cardiovascular disease, and a host of other problems. Oxidation of protein structures can alter activity, inhibit normal protein interactions, modify amino acid side chains, cleave peptide bonds, and even cause crosslinks to form between proteins. 

Formaldehyde also can be used to study protein interactions in cells or tissue sections by crosslinking and capturing protein complexes. Chapter 28, Section 1.3 describes this method and contains a protocol for use. 

Chattopadhyay et al., 1992), and a comparison of radiolabeling techniques for the crosslinker (Shephard et al., 1988). Other studies have involved the investigation of protein interactions using the label transfer nature of radioiodinated SASD (Gupta et al., 2005 Lindersson et al., 2005 LeFebvre et al., 2006). 

SADP or sulfo-SADP also have been used to study the phenylalanine-methionine-arginine-phenylalanine-amide-activated sodium channel (Coscoy et al., 1998), various apolipoprotein E isoforms (Mann et al., 1995), the high-affinity phenylalkylamine Ca2+ antagonist binding protein from guinea pig (Moebius et al., 1994), the interaction of non-histone proteins with nucleosome core particles (Reeves and Nissen, 1993), and the interactions among cytochromes P-450 in the endoplasmic reticulum (Alston et al., 1991). See Chapter 28 for methods of using photoreactive heterobifunctional crosslinkers to study protein interactions. 

Isolation of complexed molecules may be done by affinity chromatography using a column of immobilized avidin or immobilized streptavidin. Cleavage of the disulfide bond of the crosslinker may be done by treatment with 50 mM dithiothreitol (DTT). For additional information on the use of sulfo-SBED in the study of protein interactions, see Chapter 28, Section 3.1. 

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. 

Figure 18.1 A trifunctional reagent for studying protein interactions by mass spec. The bis-NHS ester arms crosslink interacting proteins, while the discrete PEG-containing biotin arm can be used to isolate or detect the conjugates using (strept)avidin reagents.

NHS-ester compounds to study protein interactions. These bis-NHS-PEG compounds may provide a superior crosslinker for studying such interactions due to their water solubility and the fact that the PEG bridge won t get buried in hydrophobic pockets on proteins or within hydrophobic membrane structures. 

BM(PEG)2 also can be used to determine protein-protein interactions or subunit interactions if there are available free thiol groups on each protein or subunit. The following protocol can be used to crosslink two thiol-containing proteins. 

Figure 28.2 DSS can capture protein interacting partners through amide bond crosslinks.

Add an aliquot of the DSS or BS3 solution to the reaction medium to obtain a final concentration of 0.5-5mM. Note Simons et al. (1999) successfully used a concentration of 0.5 mM BS3 with Madin-Darby canine kidney (MDCK) cells permanently expressing a GPI-anchored form of growth hormone decay accelerating factor (GH-DAF) to crosslink the protein interaction complexes on the cell surfaces. 

Figure 28.3 The homobifunctional crosslinkers BS2G and BS3 can be used to capture protein interactions through amide bond formation. The deuterium-labeled analogs of these reagents can be used to differentiate...

Figure 28.4 Formaldehyde can be used to capture protein interactions if it is used at low concentrations. The reaction proceeds through modification of a protein to create an intermediate immonium cation, which then goes on to react with a neighboring protein to form the crosslinked product via secondary amine bonds.

The use of PIR compounds to study protein interactions is a significant advance over the use of standard homobifunctional crosslinkers. The unique design of the PIR reagent facilitates deconvolution of putative protein interaction complexes through a simplified mass spec analysis. The software can ignore all irrelevant peak data and just focus analysis on the two labeled peptide peaks, which accompany the reporter signal of appropriate mass. This greatly simplifies the bioinformatics of data analysis and provides definitive conformation of protein-protein crosslinks. 

The following protocol is designed for treating cells with the PIR reagent to study protein interactions in vivo. It is based on the method of Tang et al. (2005). The use of the PIR compound to treat intact cells results in the crosslinking of proteins both on the cell surface and within the cell, which indicates that the reagent is able to cross the cell membrane. 

The following sections discuss the application of several photoreactive heterobifunctional crosslinkers to the study of protein interactions. 

A general protocol that can serve as a guide for the use of heterobifunctional crosslinkers in the study of protein interactions is given below. Some optimization of concentrations may have to be done depending on the particular type and properties of proteins being studied. 

Add a quantity of the Sulfo-SBED solution to the bait protein solution so that a 1- to 5-fold molar excess of crosslinker over the bait protein results in the reaction mixture. Mix well. Using greater quantities of Sulfo-SBED to the bait protein may result in precipitation due to the hydrophobic nature of crosslinker. In addition, over modification of the bait protein with the crosslinker may block sites of protein interaction, thus preventing complex formation. As a practical example, Horney et al. (2001), used a 1 1 molar ratio of Sulfo-SBED to the bait protein IGF-1 with success. 

---