Proteins
Proteins

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Proteins

7.3.1 Physical methods for demonstrating an interaction between proteins

To identify those unknown proteins in a complex mixture that interact with a particular protein of interest, protein affinity chromatography can be used (Figure 49a). This approach uses a ‘bait’ protein attached to a matrix. When this baited matrix material is then exposed to a mixture of proteins, only proteins that interact with the bait are ‘pulled out’ from the mixture. Typically, this mixture is actually an extract prepared from cells or tissue and it is passed through a chromatography column packed with inert beads that carry the bait. Proteins that interact with the bait protein are retained by the matrix and can be eluted (washed off) and then analysed. Elution may be achieved by changing the salt concentration or the pH or alternatively free bait protein could be used to displace the bound proteins. Protein microarrays (see Table 7, in next section) can be used as a high-throughput version of protein affinity chromatography.

Figure 49 Capture of interacting proteins using a bait protein in (a) protein affinity chromatography and (b) co-immunoprecipitation.
  • What requirement might make protein affinity chromatography a difficult procedure to use in practice?

  • The necessity for a purified preparation of the bait protein and selective coupling of this protein to the matrix makes this procedure technically demanding.

The same principle is used in co-immunoprecipitation (Figure 49b). This technique uses an antibody that specifically recognises the bait protein. The antibody is incubated with, for example, cell extract and binds to the bait protein. Beads coated with a protein that binds to immunoglobulins are used to pull the antibody out of the mixture, functioning in the same way as the affinity matrix in protein affinity chromatography. The beads can be spun down in a centrifuge. Along with antibody, the bait protein and the proteins that interact with the bait are precipitated in a complex. Co-immunoprecipitation has a distinct advantage over cross-linking of proteins (described below) in that the proteins are not modified in any way. Preservation of the protein in its native state is an important consideration for further analysis of its function.

  • Co-immunoprecipitation is much more versatile and is used much more widely than is affinity chromatography. Why do you think this might be?

  • Affinity chromatography requires a source of purified bait protein and selective attachment of this protein to the beads. Co-immunoprecipitation uses beads that capture all immunoglobulins and they can be used with any immunoglobulin.

Having effectively isolated binding partners for a particular protein, how can we identify or characterise them? In practice, characterisation of a protein involves a number of different experimental approaches. One of the most convenient ways of learning something about the protein is to analyse it by SDS–polyacrylamide gel electrophoresis (SDS–PAGE). From SDS–PAGE it can be determined whether a protein of interest has more than one subunit and the Mr of the component subunits can be estimated. An accurate determination of the mass of the protein can be obtained from mass spectrometry. Matrix-associated laser desorption ionisation-time-off-light spectrometry (MALDI-TOF) determines the mass and charge of peptides derived from a protein of interest. It is possible to fragment individual peptides from a protein, breaking peptide bonds, and from the differences in the masses of the products, the sequence of the peptide can be deduced. Partial sequence information can then be used to search databases to identify a match or homologous proteins.

Apart from confirming that two proteins do in fact interact, it is important to characterise their interaction. Chemical cross-linking of interacting proteins uses reagents that react with specific amino acid side-chains to covalently link those parts of two proteins that are close together. The range over which cross-linking can occur is determined by the length of the cross-linking reagent. This technique can not only help determine if two proteins do interact, but can also give information on which parts of the two proteins participate in the interaction.

A particularly powerful method for studying protein–protein interactions is surface plasmon resonance (SPR). In SPR, the bait protein is attached to a special ‘biosensor chip’ consisting of a very thin layer of metal on top of a glass prism (Figure 50). The bait is immobilised on a dextran polymer on the surface of the metal film and this surface is exposed to a flow of an aqueous solution (the mobile phase) containing a protein that is thought to interact with the bait.

Figure 50 Surface plasmon resonance. (a) When a protein in the mobile phase binds to the immobilised bait protein, there is a change in the refractive index at the surface of the metal film and a consequent change in the intensity of the reflected beam of light. (b) These changes can be monitored (over a period of minutes) and interpreted in terms of the association and dissociation of the proteins.

A light beam is passed through the prism and is reflected off the metal film, but some of the light energy is transferred to ‘packets’ of electrons called plasmons, on the surface of the metal film. This effect reduces the intensity of the reflected light and is dependent on the precise angle of incidence of the light beam, defined as the resonance angle (θ in Figure 50). The resonance angle is in turn determined by the refractive index of the solution up to 300 nm away from the metal film on which the bait protein is immobilised. (The refractive index of a material is a measure of the velocity of light through it.) If the protein in the mobile phase binds to the bait protein, the local refractive index changes, leading to a change in resonance angle. All proteins have the same refractive index and there is a linear correlation between the change in resonance angle and the concentration of protein near the surface. Thus it is possible to determine changes in protein concentration at the surface that are due to protein–protein binding. The value of this method is that the kinetics of the binding interaction can be followed in real time by analysing the rate of change of the signal. The rate of change of the signal on addition of the potential binding partner to the mobile phase is an indication of the association kinetics for the two proteins, and washing the interacting protein off the bait allows similar analysis of dissociation kinetics. Apart from protein–protein interactions, SPR is used to study many other interactions including those between proteins and DNA, carbohydrates or small ligands.

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