Protein Interaction Reporters

So far ten catalytically active caspases have been reported in mouse (caspase-1, -2, -3, -6, -7, -8, -9, -11, -12,-14) and eleven in human (caspase-1, -2, -3, -4, -5, -6, -7, -8, -9, -10, -14) (Fig. 1). Caspases are expressed as inactive proenzymes that contain an amino-terminal prodomain of variable length followed by two domains with conserved sequences a large subunit ( 20 kDa, p20) and a small carboxy-terminal subunit ( 10 kDa, plO). Caspases can be divided according to absence (-3, -6, -7, -14) or presence (-1, -2, -8, -9, -10, -11, -12) of an extended prodomain containing protein-protein interaction motifs belonging to the death domain (DD) superfamily, in particular the death effector domains (DED) and the caspase activation and recruitment domains (CARD). 

Although biologically active helical y-peptides have not been reported so far, the striking structural similarities (polarity and helicity) between the a-helix of L-a-peptides and the (P)-2.6i4-hehx of y-peptides suggest that the 2.614-helical backbone might prove useful as a template for elaborating functional mimetics of a-helical surfaces and intervening in protein-protein interactions. 

TAFs. It is conceivable that different combinations of TAFs with TBP—or one of several recently discovered TBP-like factors (TLFs)—may bind to different promoters, and recent reports suggest that this may account for selective activation noted in various promoters and for the different strengths of certain promoters. TAFs, since they are required for the action of activators, are often called coactivators. There are thus three classes of transcription factors involved in the regulation of class II genes basal factors, coactivators, and activator-repressors (Table 37-A). How these classes of proteins interact to govern both the site and frequency of transcription is a question of central importance. 

Covalent attachment of enzymes to surfaces is often intuitively perceived as being more reliable than direct adsorption, but multisite physical interactions can in fact yield a comparably strong and stable union, as demonstrated by several biological examples. The biotin/streptavidin interaction requires a force of about 0.3 nN to be severed [Lee et al., 2007], and protein/protein interactions typically require 0.1 nN to break, but values over 1 nN have also been reported [Weisel et al., 2003]. These forces are comparable to those required to mpture weaker chemical bonds such as the gold-thiolate bond (1 nN for an alkanethiol, and even only 0.3 nN for a 1,3-aUcanedithiol [Langry et al., 2005]) and the poly(His)-Ni(NTA) bond (0.24 nN, [Levy and Maaloum, 2005]). 

Figure 5.2. High-throughput mating assay for two-hybrid protein interaction screening. Yeast strains containing individual bait and prey clones are combined in a well and allowed to mate. Diploids are then selected and scored for a protein-protein interaction using the selection provided by the transcriptional reporter gene.

Figure 5.9. Split ubiquitin as a sensor for protein-protein interactions. Protein A is fused to the N-terminal domain and protein B is fused to the C-terminal domain of ubiquitin. Interaction of A and B reconstitutes a full-sized, folded ubiqutin. The folded ubiquitin is recognized by a specific protease and cleavage releases the reporter protein.

The lifetime of the excited state of fluorophores may be altered by physical and biochemical properties of its environment. Fluorescence lifetime imaging microscopy (FLIM) is thus a powerful analytical tool for the quantitative mapping of fluorescent molecules that reports, for instance, on local ion concentration, pH, and viscosity, the fluorescence lifetime of a donor fluorophore, Forster resonance energy transfer can be also imaged by FLIM. This provides a robust method for mapping protein-protein interactions and for probing the complexity of molecular interaction networks. 

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. 

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