Site-directed labeling

ASBA has been used to identify parasite adhesive proteins (Gowda et al., 2007), for active site-directed labeling of glucosidase I (Romaniouk et al., 2004), and to study interactions with the proteasome (Qureshi et al., 2003). 

Romaniouk, A.V., Silva, A., Feng, J., and Vijay, I.K. (2004) Synthesis of a novel photoaffinity derivative of 1-deoxynojirimycin for active site-directed labeling of I. Glycobiology 14, 301-310. 

B. Packard and M. Edidin, Site-directed labeling of a monoclonal antibody Targeting to a disulfide bond, Biochemistry 25, 3548-3552 (1986). 

The internal motion of T4 lysozyme in the crystal was interpreted as an inter-domain motion corresponding to opening and closing of the active site cleft (Weaver et al., 1989). Hinge-bending and substrate-induced conformational transition in T4 lysozyme in solution were confirmed in a study by site-directed labelling (Mchaourban et al., 1997). 

Figure 13 ABPP probes for metalloproteases. (a) First generation of hydroxamate-based probes with rhodamine (Rh) and Gy3 as fluorescent tags and photocross-linkers for covalent modification, (b) Active site-directed labeling mechanism for a clickable second-generation probe. (c)The potent SAHA inhibitor for HDAC classes I and II and its derived ABPP probe.

Fourier transform infrared difference spectroscopy has enormous potential. It represents a nondestructive method working at the molecular level, so the amount of material required is relatively small (40-100 fig). This enables its combination with other techniques in molecular biology. More theoretical and experimental advances are indeed possible. A theoretical means for interpreting the observed small changes of the amide I bands needs to be developed. The practical realization of the method of site-directed labeling (i.e., the incorporation of one labeled amino acid at a specific site) would represent a major breakthrough. 

Karlsson, M. Carlsson, U. Protein adsorption orientation in the light of fluorescent probes mapping of the interaction between site-directly labeled human carboiuc anhydrase n and silica nanoparticles. Biophys. J., 2005, 55(5), 3536 3544. 

Paracatalytic enzyme reactions on their part may entail paracatalytic enzyme modifications. This new mode of catalysis-linked modification is due to intramolecular reactions of unusual metastable intermediates formed during the paracatalytic reactions. It appears to afford an approach to specific active-site-directed labeling of certain enzymes. The present article describes the principal features of paracatalytic enzyme modification accompanying the oxidation of enzyme-substrate carbanion intermediates, discusses its differentiation from other substrate-dependent and catalysis-linked modifications, and concludes with an account of detailed modification procedures for several enzymes. 

In the case of the acetylcholine receptor, the recent availability of some active-site directed labelling compounds, raises some hope that its isolation will finally be accomplished, although it seems likely that more highly specific—and preferably stereospecific—compounds than those used so far will be required for this purpose. 

In the previous sections, we focused on the heme pocket of the heme proteins, whereby the intrinsic paramagnetic centers were used as the probes for structural information. However, the introduction of site-directed spin labeling has revolutionized the world of EPR in the sense that now also intrinsic diamagnetic proteins can be studied by EPR [77,78]. Indeed, in site-directed labeling, a paramagnetic spin label is attached to the protein through reaction of its functional group with the... 

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