Photoaffinity labeling studies

The location of the binding site of taxol on tubulin has been determined by photoaffinity labeling using several differently labeled taxol analogs. An early study showed that taxol labeled -tubulin as opposed to a-tubulin 466), and this result was confirmed with the azidophenyl taxol analog 15.1.1 467). 

More detailed studies showed that 3 -(p-azidobenzamido)taxol (11.2.1) photolabeled -tubulin preferentially 387) and that the site of labeling was in the N-terminal 31 amino acid unit 386). On the other hand, 2- m-azidobenzoyl)taxol (11.2.10) labeled a peptide containing amino acids 217-231 of -tubulin 393). The photoaffinity probe 11.2.5 was also found to bind (most probably) to /3-tubulin 389), while the probe 11.2.6 was shown to bind to Arg in -tubulin 468). 

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Biological Signal Transduction Basics and Opportunities for Photoaffinity Labeling Studies... 

To date the evidence seems to favor the binding of tumor promoter to phospholipid in the cell membrane. Specific binding of [3h]TPA to mouse epidermal particulate matter is susceptible to phospholipases C and A2, less susceptible to protease, and completely resistant to glycosidase (32). Photoaffinity labelling studies with [20-3h]-phorbol 12-p-azidobenzoate 13-benzoate indicates that the irreversible binding of this photolabile phorbol ester to mouse brain membrane is predominantly to the phospholipid (specifically phosphatidylethanolamine and phosphatidylserine) portion rather than to the protein portion (33). 

VI. PHOTOAFFINITY LABELING STUDIES PROBING THE MICROTUBULE AND P-GLYCOPROTEIN BINDING... 

What do SNMPs do The identification of Apo/SNMPl followed photoaffinity labeling studies that tentatively identified a 69 kDa protein as a pheromone receptor (Vogt et al., 1987) however, a role as pheromone receptor seems highly unlikely because SNMPs appear to associate with most olfactory neurons, and are neither 7-transmembrane domain receptors nor show the diversity expected for ORs. SNMPs certainly show no similarity to the presumed ORs identified in D. melangaster, A. gambiaea and H. virescens (Clyne et al., 1999 Vosshall et al., 1999 Hill et al., 2002 Krieger et al., 2002). If SNMPs are not ORs, what are they ... 

After the first photoaffinity labelling studies, which were the first methods used to define the paclitaxel binding site on tubulin, and indeed allowed identification of different amino acids of p-tubulin as putative parts of the binding site [51], the initial efforts to correlate activity (or binding) with the solid state, solution or modelled conformations of MSA in their free states led to the first pharmacophore proposals... 

Recently, we proposed a new bioactive conformation of paclitaxel, RKDOR-Taxol [50], based on (i) the 19F-13C distances obtained by the REDOR experiment [49], (ii) the photoaffinity labeling of microtubules [51], (iii) the crystal structure (PDB code 1TUB) of the Zn2+-stabilized aP-tubulin dimer model determined by cryo-electron microscopy (cryo-EM) [52], and (iv) molecular modeling (Monte Carlo Macromodel) [50], In this computational biology analysis, we first docked a paclitaxel-photoaffinity label molecule to the position identified by our photoaffinity labeling study and then optimized the... 

Esters and thioesters of (dansyldiazomethyl)methylphosphinic acid undergo carbene insertion reactions in high yield on irradiation their fluorescent properties make them suitable reagents for photoaffinity labelling studies. 

Derivatives of Cgo for photoaffinity labelling studies have been synthesized, including the cis and treats diazirine compounds (21a). The photochemical properties of these are currently being studied in various applications. An... 

The related analog 11,2.9 was also prepared in both deuterium and tritium labeled forms, but its bioactivity was such that it was not the ideal derivative for photoaffinity labeling studies (392). 

Biochemical characterization of thyroid hormone-binding species from various tissue sources revealed these to be proteins with high-affinity for thyroid hormones which are found predominantly in cell nuclei (14-16). Purification of the thyroid hormone receptor protein has been hampered by its low abundance in cells, but biochemical analysis and photoaffinity labelling studies indicate that two nuclear polypeptides of 57 and 47 kD specifically bind (17-20). 

Transfer RNA is the macromolecule which has most often been used as the label-carrying ligand in affinity and photoaffinity labeling studies of ribosomes. This is the result of the intimate involvement of tRNA in nearly all aspects of ribosomal function and the availability of convenient sites on the tRNA for the attachment of reactive groups. To date, the amino acid and the 4-thiouridine base (present in some tRNAs from Escherichia coli) have been the preferred modification sites, simply because the chemistry is straightforward and side reactions are minimal. 

Three cholesterol molecules could be docked on each AQiR subunit, rendering a total of 15 cholesterol molecules per nAChR molecule. In full agreement with the results of photoaffinity labeling studies, Baier et al. found specific cholesterol-binding sites in transmembrane domains TMl, TM3, and TM4. Interestingly, once boimd to TMl, cholesterol could not interact with TM2 due to steric hindrance. Molecular models of nAchR in complex with 15 cholesterol molecules are shown in Fig. 7.2. 

N.P. Gritsan, A.A. Koshkin, A.Yu. Denisov, Yu.Ya. Markushin, E.V. Cherepanova, A.V. Lebedev, Azidoethidium derivatives as photoaffinity labels study of the photochemistry of the monoazidoethidimn derivatives in water, J. Photochem. Photobiol. B Biol, 1997, 37, 40-51. 

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