Ligand binding site photoaffinity labeling

The not infrequent failure of the extent of attachment of photoaffinity labels to parallel receptor inactivation can be explained in several ways. First, in some membrane-bound receptor systems, receptor subunits are present in excess over the other components of the system. Second, the ligand might induce or take part in a photochemical reaction at the ligand binding site that does not culminate in covalent attachment. Ligand sensitized photooxidation can be prevented by irradiating the sample in the... 

Another test for specific labeling is to determine whether the ligand binding site is blocked. But, as 1 pointed out in the discussion of photoinactivation experiments, there are several other possible causes of apparent binding site occupation besides the covalent attachment of a ligand. It has also been noted that specific labeling as defined by a protection experiment may not always yield a blocked receptor when the photoaffinity label is a macromolecule. When sodium channels in tissue culture cells were labeled... 

Mills, J. S., Miettinen, H. M., Barnidge, D., Vlases, M. J., Wimer-Mackin, S., Dratz, E. A., Sunner, J., andjesaitis, A. J. (1998). Identification of a ligand binding site in the human neutrophil formyl peptide receptor using a site-specific fluorescent photoaffinity label and mass spectrometry./. Biol. Chem. 273, 10428-10435. 

Photoaffinity labelling is another approach to access and characterize the ligand-binding site environment in the receptor. The same group constructed two promising azido-substituted photoaffinity probes, [ IJbenzoylneonicotinoid (34) [36] and... 

Despite these difficulties, particular residues at ligand binding sites have been successfully identified by photoaffinity labeling. 

Specific reactive groups suitable for affinity and photoaffinity labeling are discussed elsewhere in this volume and in recent review articles and will not be dealt with here. It should be noted, however, that varying the length or flexibility of the reactive group may alter the reaction pattern. This approach can be used in a multicomponent system to locate components that are different distances from the ligand binding site. ... 

The key observation in affinity labeling experiments with ribosomes is that the ligand analog reacts with the receptor with a very low efficiency (see the table). This is crucial because the ribosomes are heterogeneous with respect to composition, activity, and ligand binding sites. Therefore, uncertainty exists as to which subpopulation of ribosomes actually reacts with a particular affinity or photoaffinity label. This situation is likely to exist, at least initially, in any multicomponent receptor system. 

Fig. 3.26. The interaction of macromolecular photoaffinity reagents with a receptor, a The natural ligand binds tightly to a receptor on a membrane (or in solution), b A ligand modified with a bifunctional reagent close to its site of interaction with the receptor may not bind well. c If the ligand is modified with a short-armed reagent at an alternative site it may not label the receptor but, d if a long-armed reagent is used neighboring or irrelevant polypeptides may...

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