A Powerful Tool for Life Science Research Nucleotides and nucleotide binding proteins regulate numerous biochemical processes in the cell. Many of the nucleotide binding proteins controlling these biochemical process are are the targets of drugs as well as toxicants. In addition, many of these nucleotide binding proteins are affected or altered in various disease states. The use of nucleotide photoaffinity analogs allows for the study of protein-nucleotide interactions at the molecular level. This powerful technique allows one to identify the amino acid residues actually involved in formation of the active site of nucleotide binding proteins and how mutations in that active site subsequently affect nucleotide binding. It also allows one to study how substrates, cofactors, drugs, toxicants, etc. all affect binding of nucleotides to the active site of proteins. The information gained from these studies allows researchers to determine how these nucleotide binding proteins regulate the biochemical processes in which they are involved. In addition, this technique can be used to determine which specific nucleotide binding proteins are the targets of drugs and toxicants as well as how these proteins are regulated. Background Nucleotide photoaffinity analogs such as 8-Azido ATP (8N3ATP, products # AT02, AP02, AP03) and 8-Azido GTP (8N3GTP, products # AT04, AP04, AP05) are powerful tools for such studies. These analogs contain a photoactive azido (-N3) group substitution in the base ring of the nucleotide. The azido group is chemically inert until photoactivated by UV light. Upon photolysis it generates a highly reactive nitrene which inserts into either the peptide backbone or the amino acid side chains of the protein to which it is bound. This insertion forms a covalent linkage between the photoprobe and the protein allowing it to be permanently tagged for identification. The illustration below shows the formation of the nitrene upon photolysis of 8N3ATP . The destruction of the azide upon photolysis can be easily monitored by recording the UV spectra before and after photolysis. Figure 1
Another photoactive moiety which can be easily attached to nucleotides is the benzophenone group. Benzophenone amine can be directly coupled to ATP through the 5' gamma phosphate to give ATP[g]BP (products # AT07, AP07). In contrast to the azido group, benzophenone is activated by long-wave length UV light (320-360nm). Upon exposure to long-wave length UV light, the benzophenone group's double-bonded oxygen forms a triplet biradical that can abstract hydrogen from the protein to which the nucleotide is bound, ultimately causing the two molecules to be covalently linked. Figure 2
Photoaffinity vs Chemical Affinity Labeling Photoaffinity labeling differs in several ways from classical chemical affinity labeling. Chemically reactive probes normally have an electrophillic group which reacts with basic amino acids such as arginine or lysine or with sulfhydryls containing amino acids such as cysteine. Although this method results in higher stoichiometry of labeling, a large proportion of it can be nonspecific (outside the active site). This is due to the ability of chemically reactive probes to potentially react with all these available amino residues which are accessible not only those just within the active site. In contrast, photoaffinity probes have a chemically inert photactive azido group. This distinctive feature allows the study of enzymatic processes without the destruction of the azido group. The optimal bound analog in the enzyme active site can then be photoactivated by brief exposure to UV light. The reactive intermediate has a short half life and reacts indiscriminately to form a covalent bond with amino acid residues within the active site. The Photolabeling Procedure The photolabeling procedure involves initial incubation of the radiolabeled photoprobe with the protein or a crude mixture of proteins. (i.e. cell lysate or tissue homogenate). Brief (1-2 min.) photolysis with a low intensity (2000-6000 µW), hand held 254 nm UV light produces a covalent linkage within the active site. Immediately after photolysis, the photolabeling reaction is quenched with a thiol reagent such as dithiothreitol or beta mercaptoethanol (10 mM or greater). The photolabeled protein is then separated from the unbound photolyzed probe by several methods such as precipitation with acid, ammonium sulfate, polyethylene glycol, etc. or by gel filtration or ultrafiltration. The photolabeled proteins can then be analyzed by virtually any biochemical method: SDS-PAGE, two dimensional IEF x SDS-PAGE, FPLC, HPLC etc. Quantification of the degree of photoincorporation of radioactivity can then be simply determined by any of a number of commonly used techniques such as autoradiography, imaging analysis or scintillation counting. Advantages of Photolabeling Nucleotide photoaffinity analogs such as 8-N3ATP and 8-N3GTP can provide a wealth of information regarding the biochemical properties of nucleotide binding proteins. Elucidating the structure of the active site is the one of major objective of such studies. With structural isomers 8-N3ATP (syn conformation) and 2-N3ATP (anti conformation) a complete understanding of the active site is possible (see 2-N3ATP Tautomers). ALT offers several photoprobes both unlabeled and labeled with 32P that are intended for this purpose. The major benefits of these powerful research tools include simple experimental setup and remarkable specificity as illustrated below. Figure 3
Photoaffinity labeling of Creatine Kinase (CK) with [g32P] 8-N3ATP illustrating the specificity of nucleotide photoaffinity analogs. CK, a known ATP binding protein was photolabeled with [g32P] 8-N3ATP in the absence of any competitor (lane 1), in the presence of 10-fold excess ATP (lane 2) and in the presence of 10-fold excess GTP (lane 3). The autoradiogram shows that ATP was effective in preventing photoinsertion by the photoprobe and GTP was not. Words of Caution Reducing Agents Photoactive azides (-N3) are extremely susceptible to reduction to non-photoactive amines (-NH2) by most commonly used reducing agents including dithiothreitol (DTT) and ß-mercaptoethanol (ßME). Therefore, these reducing agents should omitted from the the photolabeling buffer if at all possible. Often times DTT and ßME are added to purified proteins or homogenates to keep any oxidation sensitive enzymes in a fully reduced and active state. In these cases, we have found that if the reducing agent can be diluted to a final test concentration of less than 50 µM for DTT or less than 100 µM for ßME there is little loss of photoactivity. If however, the concentration of the reducing agent can not be diluted to these levels, it is best to remove or dilute them immediately before photolabeling the sample. One of the easiest, most effective and least time consuming methods of accomplishing this is by ultrafiltration. We have used Amicon Microcon and Centricon ultrafiltration devices with very good results. Using these devices the protein or homogenate can be washed free of reducing agents and then resuspended in the appropriate buffer immediately before photolabeling. We have found that even the most oxidation sensitive enzymes exhibit very little loss of activity using this procedure. Technical Information or General Inquiries Contact Dr.
Anjan Bhattacharyya, Ph.D.
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R
= ATP or GTP