Binding partner

PTKs can be subdivided into two large families, receptor tyrosine kinases (RTKs) and non-RTKs. The human genome encodes for a total of 90 tyrosine kinases of which 32 are nonreceptor PTKs that can be placed in 10 subfamilies (Fig. 1). All nonreceptor PTKs share a common kinase domain and usually contain several additional domains that mediate interactions with protein-binding partners, membrane lipids, or DNA (Table 1). These interactions may affect cellular localization and the activation status of the kinase or attract substrate proteins for phosphorylation reactions. 

For a soluble enzyme that is not part of a multi-enzyme complex, the fastest rate of enzyme-inhibitor association is determined by the rate of molecular collisions between the two binding partners (i.e., the enzyme and the inhibitor) in solution. The rate of molecular collisions is in turn controlled by the rate of diffusion. The diffusion-limited rate of molecular collisions is dependent on the radii of the two binding molecules and the solution temperature and viscosity (Fersht, 1999) ... 

Figure 7.9. Schematic diagram of a surface plasmon resonance biosensor. One of the binding partners is immobilized on the sensor surface. With the BIACORE instrument, the soluble molecule is allowed to flow over the immobilized molecule. Binding of the soluble molecule results in a change in the refractive index of the solvent near the surface of the sensor chip. The magnitude of the shift in refractive index is related quantitatively to the amount of the soluble molecule that is bound.

The experiments described above indicate that technology is available to couple SPR with mass spectrometry. These methods should be useful for protein-protein interaction mapping. For example, immobilized proteins can be used as hooks for fishing binding partners from complex protein mixtures under native conditions. The coupling of techniques can lead not only to the rapid identification of interacting proteins but will also provide information on the kinetic parameters of the interaction. This approach should serve as an excellent complement to the use of in vivo techniques such as the yeast two-hybrid system. 

Table 1 Readers of methyl lysine marks and representative binding partners...

To provide the reader with a more complete picture, this section describes the use of affinity tags other than a Hisg-tag which were also used successfully for purification of eIF3 and its binding partners (Asano et ah, 2000 Valasek et ah, 2003). 

Of the large number of protein interactions that take place in cells, perhaps the vast majority may be described as transient. Most proteins that modify other molecules do so very rapidly and so interact only briefly with their substrates or binding partners (i.e., enzymes). In addition, since proteins within cells are highly compartmentalized, the affinity of most interactions doesn t have to be very great, because each potential binding partner is within short diffusion distances and the relative concentration of molecules within these small volumes is high. 

FIGURE 7-4 Basic cadherin structure. Two types of cadherin are present in the nervous system the classic cadherins with five extracellular domains and the protocadherins with six extracellular domains. For the classic cadherins, the binding partners on the cytoplasmic side include the a, P and y catenins. The binding partners for the protocadherins have not yet been identified. 

A simple method to analyze binding equilibria with slow association and dissociation kinetics is the precipitation of one binding partner with an antibody or... 

Because the precipitation (or affinity binding step) perturbs the equilibrium of interest it has to be ensured that removal of one reactant is much faster than the dissociation or association of the binding partners. 

Figure 11A shows a theoretical example of a titration curve A + B = AB, where the signal is proportional to the amount of complex. The solid lines represent conditions where Bmax is equal to KD. Here for both presentations of signal vs either [Atotal] (total concentration of A added to the preparation) or [Afree] (concentration of non-complexed A in the solution, calculated as [Atotal] - ([AB]) the plot is curved and allows discrimination between free and complexed binding partners. If [Bmax] is substantially higher than KD the issue of active site... 

Determination of three-dimensional protein structures has become an important tool in protein research, indicated by the exponentially growing number of protein data bank entries during the last decade. They have contributed considerably to our understanding of function and mechanism of biomolecules. Structures of proteins alone or in complex with their substrates or binding partners like cofactors, DNA, or other proteins can visualize interactions at the atom-... 

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