A photogenerated pore-forming protein

Chang, Chih-Yuan; Niblack, Brett; Walker, Barbara; Bayley, Hagan

doi:10.1016/1074-5521(95)90220-1

Cited by 106 publications

(83 citation statements)

References 46 publications

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“…Indeed, after this work was complete, the data were used to guide the construction of a photoactivatable ␣HL by targeted chemical modification of cysteine (37). Cysteine residues might also be used in the construction of hybrid proteins or for attaching the pores to surfaces.…”

Section: Discussionmentioning

confidence: 99%

Key Residues for Membrane Binding, Oligomerization, and Pore Forming Activity of Staphylococcal α-Hemolysin Identified by Cysteine Scanning Mutagenesis and Targeted Chemical Modification

Walker

Bayley

1995

Journal of Biological Chemistry

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165

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Section: Discussionmentioning

confidence: 99%

Key Residues for Membrane Binding, Oligomerization, and Pore Forming Activity of Staphylococcal α-Hemolysin Identified by Cysteine Scanning Mutagenesis and Targeted Chemical Modification

Walker

Bayley

1995

Journal of Biological Chemistry

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150

165

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“…Large quantities and many varieties of precisely defined caged peptides and, by use of peptide ligation techniques, caged proteins can be prepared by this strategy. Drawbacks of earlier methods used to prepare caged proteins (26)(27)(28) include that incorporation of photolabile groups often occurs with varying efficiency into multiple sites, and photolytic conversions are low. Selective incorporation of caged amino acids into proteins by in vitro translation has been achieved (29), but caged proteins were obtained in limited quantities.…”

Section: Discussionmentioning

confidence: 99%

Signaling pathways underlying eosinophil cell motility revealed by using caged peptides

Walker

Gilbert

Drummond

et al. 1998

Proc. Natl. Acad. Sci. U.S.A.

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Insights into structure-function relations of many proteins opens the possibility of engineering peptides to selectively interfere with a protein's activity. To facilitate the use of peptides as probes of cellular processes, we have developed caged peptides whose inf luence on specific proteins can be suddenly and uniformly changed by near-UV light. Two peptides are described which, on photolysis of a caging moiety, block the action of calcium-calmodulin or myosin light chain kinase (MLCK). The efficacy of theses peptides is demonstrated in vitro and in vivo by determining their effect before and after photolysis on activities of isolated enzymes and cellular functions known to depend on calcium-calmodulin and MLCK. These caged peptides each were injected into motile, polarized eosinophils, and when exposed to light promptly blocked cell locomotion in a similar manner. The results indicate that the action of calcium-calmodulin and MLCK, and by inference myosin II, are required for the ameboid locomotion of these cells. This methodology provides a powerful means for assessing the role of these and other proteins in a wide range of spatio-temporally complex functions in intact living cells.Several methods have been used in the past to probe the role of a protein in cell function, each with advantages as well as limitations. Organic compounds are available that can modulate the activity of proteins, but interpretation of effects often is complicated by their relatively slow onset and low selectivity for a specific protein. Impressive progress toward single protein specificity has been made with antisense (1) and homologous recombination (2) methods, which disrupt the expression, and thus the function of a specific protein. Interpretation of effects, or lack thereof, on a cellular function may be complicated, however, by compensatory pathways enhanced by the absence of the targeted protein. Peptides that bind to proteins with high affinity and high selectivity provide a means to rapidly and potently inhibit the activity of selected proteins, but peptides must be microinjected into cells, and microinjection itself can at least transiently alter cell function. It thus would be desirable to have a way to make a peptide that is initially inactive or ''caged'' because of a strategically placed photolabile moiety (3, 4). Such a peptide could be injected into a cell, and time allowed for it to distribute evenly and for normal cell function to be verified. The peptide's biological activity then could be unmasked by light-directed removal of the photolabile group. Each cell would be its own control, thereby diminishing effects of cell to cell variability, and active peptides could be produced rapidly (within milliseconds) and with good spatial resolution.We describe here the preparation and use of photoactivatable caged peptides targeted against calmodulin and myosin light chain kinase (MLCK). Because these two proteins are known to be essential in the control of smooth muscle contractility, the efficacy of the caged...

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“…Catalytic antibodies function like enzymes, and are created to catalyze reactions for which there are no naturally occurring enzymes [71,72]. Fifty or more reactions were made by the action of catalytic antibodies, which were obtained individually by the methods of Protein Engineering (PE) [30,[73][74][75][76] What does a vaccine contain?…”

Section: Cancer Immunotherapymentioning

confidence: 99%

Vaccines against Pathogens: A Review and Food For Thought

Amara¹

2016

SOJB

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A photogenerated pore-forming protein

Cited by 106 publications

References 46 publications

Key Residues for Membrane Binding, Oligomerization, and Pore Forming Activity of Staphylococcal α-Hemolysin Identified by Cysteine Scanning Mutagenesis and Targeted Chemical Modification

Key Residues for Membrane Binding, Oligomerization, and Pore Forming Activity of Staphylococcal α-Hemolysin Identified by Cysteine Scanning Mutagenesis and Targeted Chemical Modification

Signaling pathways underlying eosinophil cell motility revealed by using caged peptides

Vaccines against Pathogens: A Review and Food For Thought

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