Caged Peptides

The peptide was purified by analytical RP-HPLC in MeCN/H20/TFA. To generate the free a-carboxyl on the cage moiety, the purified peptide was demethylated by treatment with 10% K2C03, neutralized with 1M HC1, and repurified by RP-HPLC. The demethylated caged peptide, 9cgY-LSM-l, resolved into two HPLC peaks, which appeared to be stereoisomers. The peptide was double lyophilized to remove traces of TFA and characterized by amino acid analysis and mass spectrometry. 

Caged peptides (NPY and angiotensin II) containing Tyr[Bzl(2-N02) were synthesized on an automated solid-phase peptide synthesizer using Fmoc chemistry and cleaved from the resin by treatment with TFA. Crude peptides were purified by semipreparative HPLC with a gradient phase of 0.1% TFA and MeCN. For fractions containing peptides, the eluant was not passed through the UV monitor to avoid potential photolysis. The collected peptide was examined by analytical HPLC and MS. 

When REFs were exposed to the membrane-permeant PKA activator, 8-(4-chlorophenylthio)-cAMP (CPT-cAMP), they underwent the same morphological transformation as described above (disruption of actin stress fibers leading to cell shape changes). In contrast, cells microinjected with the caged peptide (5 pM estimated intracellular concentration) and exposed to UV irradiation were unable to respond to the CPT-cAMP stimulus, demonstrating that the CPT-cAMP activation of the PKA pathway had been efficiently blocked in vivo by the decaged peptide [152],... 

Recently, a Ser-caged, photoactivatable fluorescent peptide probe that monitors protein kinase C (PKC) activity was described [75]. As expected, the Ser-caged peptide failed to serve as an effective PKC substrate in vitro, but upon light-induced deprotection (300-400nm, A.max360nm, 90s), the serine became phosphorylated and enzyme activity was recorded as a convincing change in the fluorescent properties of the probe. Photoconversion was estimated to occur with 60% yield and a quantum yield of 0.06. 

Figure 12 Kinetic profiles of product formation in reactions between E and N in the presence of the caged peptide T in the dark (black dashed line) and after shining light (solid black line). The kinetics of ligation reactions between E and N without any template seeded (blue dashed lines) or seeded with premade T molecules (blue solid line) are shown for comparison.

Bayley H., Chang, C.-Y., Miller, W. T., Niblack, B., and Pan, P. "Caged Peptides and Proteins by Targeted Chemical Modification." Afef/iods Enzymol., 291,117 (1998). 

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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