Protein interactions captured using

New developments in immobilization surfaces have lead to the use of SPR biosensors to monitor protein interactions with lipid surfaces and membrane-associated proteins. Commercially available (BIACORE) hydrophobic and lipophilic sensor surfaces have been designed to create stable membrane surfaces. It has been shown that the hydrophobic sensor surface can be used to form a lipid monolayer (Evans and MacKenzie, 1999). This monolayer surface can be used to monitor protein-lipid interactions. For example, a biosensor was used to examine binding of Src homology 2 domain to phosphoinositides within phospholipid bilayers (Surdo et al., 1999). In addition, a lipophilic sensor surface can be used to capture liposomes and form a lipid bilayer resembling a biological membrane. 

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. 

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. 

The techniques developed to study protein interactions can be divided into a number of major categories (Table 31.1), including bioconjugation, protein interaction mapping, affinity capture, two-hybrid techniques, protein probing, and instrumental analysis (i.e., NMR, crystallography, mass spectrometry, and surface plasmon resonance). Many of these methods are dependent on the use of an initial bioconjugation step to discern key information on protein interaction partners. 

Many of the methods developed to study protein interactions use the bait/prey model to detect interacting partners (Phizicky and Fields, 1995 Archakov et al., 2003 Piehler, 2005). The bait protein is a purified protein (often recombinant) that is used to lure and capture a putative interacting protein or biomolecule. The bait protein may be immobilized to a solid phase for affinity separations or be used in solution. It also may be fusion tagged (i.e., GST or 6X His) or labeled with a detectable molecule, such as a fluorescent probe. It often is the case... 

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 following protocol is a generalized method that summarizes the publications on the use of formaldehyde for capturing interaction proteins. The ranges indicated for concentrations of reactants and time of the reaction need to be optimized for each protein interaction studied. 

Figure 28.13 A sulfo-SBED-captured protein interaction can be released using DTT to cleave the disulfide within the cross-bridge leading to the bait protein. The result transfers the biotin label to the unknown interacting protein. The biotin tag thus allows the interacting protein to be detected or isolated using (strept)avidin reagents.

Kim, M., Park, K., Jeong, E. J., Shin, Y. B., Chung, B. H. (2006) Surface plasmon resonance imaging analysis of protein-protein interactions using on-chip-expressed capture protein. Anal. Biochem. 351,298-304. 

Some arrays used in proteomics contain antibodies covalently bound onto the array surface for immobilization. Then these antibodies capture corresponding antigens from a complex mixture. Afterwards, a series of analysis are carried out. For instance, bound receptors can reveal ligands. With this information in hand, binding domains for protein-protein interactions can be detected. The main problem in using microarray methods for proteomics is that protein molecules must show folding with the array in the correct conformation during the preparation and incubation. Otherwise, protein-protein interactions do not take place. 

In addition to finding small organic molecules that bind to a protein, covalent capture methods can identify peptides that interact with proteins. Kohda and colleagues used this approach to study the mitochondrial protein Tom20, an import receptor that recognizes an epitope on proteins targeted for the mitochondria.1301 Previous work had characterized this epitope as a five-residue peptide that assumes an amphiphilic helical conformation, and coarse sequence preferences had been worked out. However, Tom20 has both low affinity... 

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