Binding site accessibility

Binding to transport proteins may be of particular interest, since binding not only assays the affinities of the binding site on the transporter protein but also the translocation equilibria [67], In terms of enzyme catalysis, a transport protein transforms a substrate, a molecule located at one side of the membrane, into a product, the same molecule at the other side of the membrane, without chemical modification. Substrate must bind to a particular conformation of the enzyme with the binding sites accessible only from, for example, the outside. Similarly, the release of the product has to occur from a conformation which opens the binding site to the inside only this implies at least one transition step between the two types of conformations (see Fig. 

Apo-IRP-1 no [4Fe-4S] cluster Cytoplasmic aconitase with [4Fe-4S] cluster IRE-binding site accessible IRE-binding site not accessible... 

IRE-binding site accessible (proteasome-mediated) degradation... 

The use of enzymatic tracers for MIP-ILAs has been limited due, on the one hand, to the poor performance of these biomolecules in organic media where many MIPs work and, on the other, to the limited accessibility of the large protein macromolecules to the binding sites of these hydrophobic and highly cross-linked materials. The use of imprinted microspheres or films with binding sites at or close to the surface can overcome the problem of binding site accessibility [9]. 

Figure 8 shows a scheme of the reaction cycle of copper ATPases, assuming that they work by a mechanism analogous to that of Ca - or Na+, K+-ATPases. To pump ions, the enzyme must cycle between a state with a high-affinity copper-binding site accessible from only one side of the membrane and a low-affinity state in which the copper cavity is accessible from the other side of the membrane. The high- and low-affinity forms of P-type ATPases were initially named Ei and E2 by Racker (1980) and for many years these ATPases were called E]E2-ATPases, until they were renamed P-type by Pedersen and Carafoli (1987a). 

The sensitivity of an optical biosensor depends on two main factors the capacity of the sensing layer to bind the analyte and the optical detection limit of the device (minimum amount of analyte able to trigger a signal). The first depends on the affinity of the interaction and the number of binding sites accessible to the analyte. The second depends on the molecular weight of the analyte, the signal-to-noise ratio and drift. 

Figure 13.2 Pump action. A simple scheme for the pumping of a molecule across a membrane. The pump interconverts to two conformational states, each with a binding site accessible to a different side of the membrane.

Overall, thermal treatments appear to uniformly alter the MIP matrix and do not target speeifie elasses of sites. The result is that the annealed MIPs show equal or lesser affinity and selectivity. In some cases, the decrease in binding properties was somewhat offset by improvements in material properties such as binding capacity, binding site accessibility, and reduction of the slow leaching of the template molecule from the matrix [13]. To date, no examples have been reported in which thermal treatments of MIPs led to selective alterations of the matrix, and this may be a fruitful area for future studies. 

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