Binding sites caveats

There is one major caveat of using the tissue culture transport experiment to study P-gp efflux that cannot be overlooked—P-gp efflux is not directly determined in this experiment. Rather, the effects of P-gp-mediated efflux activity and changes to this activity are inferred from the resulting overall transport data. Particularly with regards to substrate identification, there is the potential for false negatives. For a compound to be affected by P-gp-mediated efflux, it must reach P-gp s binding site that is within the cell. Compounds with poor membrane (transcellular) permeability are not likely to be identified as substrates (395,397). Conversely, compounds with very high passive membrane permeability can saturate P-gp efflux at low micromolar concentrations and are often not identified as substrates... 

Statistics including large and diverse sets of protein structures have revealed that the 20 amino acid residue types exhibit distinctly different levels of flexibility [138], Gin residues appear to be amazingly flexible (given their medium size) and are used in the following section for exemplifying some of the caveats that complicate binding site comparisons. 

With these caveats in mind, a number of points support a relatively short distance for guanine-Ru(bpy)3 electron transfer in our system. First, as shown in Fig. 3, when the guanines are scarce in the sequence, the binding of Ru(bpy)3 at sites on the helix to which electron transfer is not efficient must be considered [19]. Thus, the oxidant can bind to the duplex at sites too far from a guanine for electron transfer. Quantitative analysis of these data suggests that electron transfer occurs within distances that are consistent with those observed in studies of covalently bound donors and acceptors and therefore with values of the tunneling parameter fi consistent with those observed in other systems [38, 50, 98-100]. 

In one approach, the free energies of binding, out of water into the enzyme active site, of the reactant(s) and transition structure are computed, in order to see if rate acceleration can be explained by selective binding of the transition structure. However, there are several caveats associated with such an approach. First, it must be decided whether to use the same reactant and transition state structures in solution and in the enzyme. If the same structures are used, then the potential for catalysis specifically by selective transition state binding can be quantified. Of course, the actual enzyme-bound structures may be different than those in aqueous solution, and... 

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