Binding Sites. Site Control

In order to be able to form stable complexes, a ligand should interact more strongly with the cation than the solvent. The solvation energy has to be overcome by the interaction of the cation with the ligand binding sites. The choice of binding site parameters should thus allow efficient control of the complexation properties of a ligand. 

Other suitable binding sites, which in addition are chemically inert, are ether oxygens and tertiary amine nitrogens. Since most synthetic 

Anionic binding sites favour small cations over large ones and divalent cations over monovalent ones the presence or absence of such a site is expected to have a very marked effect on selectivity. 

Neutral oxygen and nitrogen sites are much to be preferred to neutral sulfur sites however, sulfur, may bind quite strongly to small, highly charged cations because of its high polarizability. 

Dispersion attractions are usually weak, increasing from small to large cations, (of the order of 0.1, 1.0 and 1.5 kcal/mol for Li+, K+ and Cs+, respectively) they slightly favour nitrogen sites over oxygen sites. 

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It is not necessarily the strength of monovalent cation binding that determines the activating ability. This was demonstrated by Di Cera and coworkers. They were able to convert thrombin from a sodium-specific enzyme to a potassium-specific enzyme. This was done by site-directed mutagenesis by redesigning a loop in the backbone that defines the geometry near the cation-binding site and controls access to it. They found, however, that, while the mutant enzyme bound K+ better than Na+, it was still preferentially activated by K+ rather than Na+. 

Salis, H.M., Mirsky, E.A., and Voigt, C.A. (2009) Automated design of synthetic ribosome binding sites to control protein expression. Nat. Biotechnol., 27, 946-950. 

It is common practice to cheek that the label is site-directed by inhibiting the covalent attachment of the affinity label (for instance, a DNP-based reagent) by an excess of a nonreactive analog, i.e., DNP-lysine. It should be stressed that, as evidence for attachment of the label to a sin e binding site, this control is by itself insufficient and is based on circular reasoning. Few, if any, affinity reagents are universally reac-... 

We are continuing this type to study using different synthetic polypeptides with the aim of finally arriving at a sort of thermodynamic mapping of the important binding sites which control native conformation and function of RNase-A. 

Figure 8.1 A region of DNA in the related bacteriophages lambda, 434, and P22 that controls the switch for synthesis of new phage particles. Two structural genes are involved in this switch one coding for a repressor protein and one coding for the Cro protein. Between these genes there is an operator region (OR) that contains three protein binding sites—ORl, OR2, and OR3.

Muscle glycogen phosphorylase is a dimer of two identical subunits (842 residues, 97.44 kD). Each subunit contains a pyridoxal phosphate cofactor, covalently linked as a Schiff base to Lys °. Each subunit contains an active site (at the center of the subunit) and an allosteric effector site near the subunit interface (Eigure 15.15). In addition, a regulatory phosphorylation site is located at Ser on each subunit. A glycogen-binding site on each subunit facilitates prior association of glycogen phosphorylase with its substrate and also exerts regulatory control on the enzymatic reaction. 

Repression of genes is associated with reversal of this process under the control of histone deacetylases (HDACs). Deacetylation of histones increases the winding of DNA round histone residues, resulting in a dense chromatin structure and reduced access of transcription factors to their binding sites, thereby leading to repressed transcription of inflammatory genes. 

The microtubule-associated proteins MAP2 and tau both have two separate functional regions (Lewis et al., 1989). One is the microtubule-binding site, which nucleates microtubule assembly and controls the rate of elongation (by slowing the rate of assembly). The second functional domain shared by MAP2 and tau is a short C-terminal a-helical sequence that can cross-link microtubules into bundles by self-interaction. This domain has some of the properties of a leucine zipper. Likely it is responsible for the organization of microtubules into dense stable parallel arrays in axons and dendrites (Lewis et al., 1989). 

This example assumes that RIA was chosen. The principle behind RIA is the competition between the analyte A and a radioactively tagged control C (e.g., a /-marked ester of the species in question) for the binding site of an antibody specifically induced and harvested for this purpose. The calibration function takes on the shape of a logistic curve that extends over about three orders of magnitude. (Cf. Fig. 4.38a.) The limit of detection is near the B/Bo = 1 point (arrow ) in the upper left corner, where the antibody s binding sites are fully sequestered by C the nearly linear center portion is preferrably used for quantitation. 

We have already mentioned the application of supercomputers to biochemical simulations. Internal dynamics may play an Important role In such simulations. An example would be enzyme binding-site fluctuations that modulate reactivity or the dynamics of antigen-antibody association (11). In the specific case of diffusion-controlled processes, molecular recognition may occur because of long-range sterlc effects which are hard to assess without very expensive simulations (12.)-... 

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