Protein-protein interactions are intrinsic to virtually every cellular process and they play an important role in cell processes. The subject of protein-protein interactions represents a vast ensemble of result from biological, biochemical and biophysical studies carried out to date and can not be treated in its entirety in any reasonable fashion (Royer, 2004).
Proteins are composed of more than one subunit are found in many different classes of proteins. Some of best-characterized multisubunit proteins are those that, as originally purified, contained two or more different components. These include classical proteins such as hemoglobin, tryptophan synthetase, aspartate transcarbamylase, core RNA polymerase, Qβ-replicase, and glycyl-tRNA synthetase. Since these proteins purified as multisubunit complexes, their protein-protein interactions were self-evident (Phizicky and Fields, 1995).
Other well-known examples of multisubunit proteins include metabolic enzymes such as the pyruvate dehydrogenase ligation complex, the DNA replication complex of E.coli and other organisms, the bacterial flagellar apparatus, the nuclear pore complex, and the tail assembly of bacteriophage T4. Also included in this group are ribonucleoprotein complexes, such as the signal recognition particle of the glycosylation pathway, small nuclear ribonucleoproteins of the spliceosome, and the ribosome itself. Although some of the subunits of these protein complexes are not tightly bound, activity is associated with a large structure that in many cases is called a protein machine (Alberts and Miake-Lye, 1992).
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There are also a large number of transient protein-protein interactions, which in turn control a large number of cellular processes. All modifications of proteins necessarily involve such transient protein-protein interactions (Morell et al., 2007). Transient protein-protein interactions are also involved in the recruitment and assembly of the transcription complex to specific promoters, the transport of proteins across membranes, the folding of native proteins catalyzed by chaperonins, individual steps of the translation cycle, and the breakdown and re-formation of subcellular structures during the cell cycle (Phizicky and Fields, 1995).
The protein-protein interactions can be detected and studied by many approaches, which can be divided into physical methods, library-based methods and genetic methods. In next chapters we will show a principle, procedure of particular methods and the possibility of their applications to study of ter determinants.
3.1. Physical methods.
3.1.1. Protein Affinity Chromatography
Affinity chromatography is a biochemical method designed to separate proteins from a mixed sample. A protein can be covalently coupled to a matrix such as Sepharose under controlled conditions and used to select ligand proteins that bind and are retained from an appropriate extract. Most proteins pass through such columns or are readily washed off under low-salt conditions; proteins that are retained can then be eluted by high-salt solutions, cofactors, chaotropic solvents, or sodium dodecyl sulfate (SDS) (Phizicky and Fields, 1995).
This technique has been used to detect interactions among replication proteins produced in T4 bacteriophage-infected E. coli (Alberts et al., 1983), to detect week protein-protein interactions among the replication complex components (Formosa et al., 1991), to detect phage and host proteins that interacted with different forms of E. coli RNA polymerase (Ratner, 1974). Protein affinity chromatography has also been useful for illuminating the nature of other complex protein machines such as those responsible for transcription in bacteriophage λ-infected E. coli and in mammalian cells (Formosa et al., 1991; Greenblatt et al.,1981; Burton et al., 1988).
The affinity chromathography is very often used as purification method for protein complexes and fused proteins (Greenblatt et al.,1981; Mayer et al., 1991; Hu et al., 1992; Weng et al., 1993; Zhang et al., 1993), or influence of modification state (Ludlow et al., 1989; Ludlow et al., 1993; Wiman, 1993), or retention of native structure of the couple protein (Ratner, 1974; Greenblatt et al., 1981; Kellogg et al.,1989).
3.1.2. Affinity Blotting
Affinity blotting is a procedure analogous to the use of affinity columns. In this technique proteins are fractionated by PAGE, thereafter transferred to a nitro-cellulose membrane, and identified by their ability to bind a protein, peptide, or other ligand. This method is similar to immunoblotting (Western blotting), which uses an antibody as the probe. Complex mixture of proteins, such as total-cell lysates, can be analysed without any purification. Cell lysates can also be fractionated before gel electrophoresis to increase the sensitivity of the method for detecting interaction with rare proteins (Phizicky and Fields, 1995).
3.1.3. Blue-Native PAGE.
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Blue-Native Polyacrylamide Gel Electrophoresis (BN-PAGE) was originally described by Schägger and von Jagow (1991) as a technique for the separation of enzymatically active membrane protein complexes under mild conditions. In this variation of gel electrophoresis, the anionic dye Coomassie Brilliant Blue is added to the sample prior to loading and binds to protein complexes during electrophoresis under physiological conditions. The technique has gained interest from researchers focused on functional proteomics in recent years, as it allows the study of protein-protein interactions (Krause, 2006).
BN-PAGE is a charge shift method, in which the electrophoretic mobility of a multi-protein complex (MPC) is determined by the negative charge of the bound Coomassie dye and the size and shape of the complex. Coomassie does not act as a detergent and preventes the structure of MPCs (Camacho-Carvajal et al., 2004).
Analysis of protein complexes is usually carried out by the combination of BN-PAGE and SDS-PAGE in a two-dimentional (2D) approach. The whole-cell lysates are first separated on BN-PAGE as first dimension. For further separation in a second-dimension SDS-PAGE, the lanes from first-dimension BN-PAGE are cut out and placed into a second-dimension SDS-PAGE of the same thickness. This procedure is illustrated in Fig 6