Photoactivatable Groups amino

This approach has been successful in a number of affinity labeling experiments. The basic protocol requires using both a pep-tRNA and an aa-tRNA. One of them has a radioactive amino acid (alternatively, the two amino acid moieties may be labeled with different radioisotopes) the other has an electrophilic or photoactivatable group attached to the side chain or the -amino nitrogen of the amino acid. The pep-tRNA is bound to ribosomes under conditions that favor binding to the P site. If this tRNA contains the affinity or photoaffinity label, reaction with the ribosomal components is allowed to proceed. Then the aa-tRNA is added to the incubation, and a peptide bond is allowed to form between the two aminoacyl moieties. Alternatively, the reactive probe may be attached to the aa-tRNA. Since peptide bond formation follows rapidly upon binding, the covalent reaction of the probe with ribosomal components presumably occurs after peptide transfer. 

The major requirements for photoactivatable amino acids are that they should exhibit high optical purity and contain appropriate protecting groups for solid-phase synthesis there is also a particular advantage if they contain the radiolabel. While p-azido-phenylalanines can easily be labeled with 125I and... 

Hydroxyl groups can be introduced by the treatment with sodium borohydride [21], The hydroxyl groups (PEEK-OH) can be further reacted with 4-amino-benzoic acid or succinamic acid to give carboxyl-modified PEEK surfaces. Moreover, PEEK can be aminated [22]. Functionalized arylazides can be readily grafted on the PEEK film surfaces by UV irradiation [23]. Aromatic azides, such as 4-azido-tetra-fluorobenzoic acid, or A-butyl-A -(4-azidophenyl)-thiourea, belong to the class of photoactivatable reagents. These compounds can be photo-grafted onto PEEK. 

Unlike most proteins, where precise control of the caging reaction is difficult to achieve, chemically synthesized peptides can be caged relatively easily at specific amino acids.In one case, this approach was used to generate photoactivatable ribonuclease S. This enzyme is composed of two units S-peptide (Residues 1 to 20) and S-protein (Residues 21 to 124). A series of S-peptides carrying a 2-nitrobenzyl caging group on either Asp, Glu, Gin, or Lys side chains was prepared by chemical synthesis and chimeric RNAse S variants composed of these caged peptides assembled. In one case, a complete inactivation of RNAse S was achieved with 37% activity recovered upon illumination. 

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