Photomodulation of the nucleating activity of a photocleavable crosslinked actin dimer.
The ability to generate substrate concentration jumps through photo-deprotection of amine, carboxyl and phosphate groups has been an important development for investigations of protein activity in complex systems. To broaden the versatility and applications of photo-deprotection techniques for the photomodulation of protein activity we describe the synthesis and characterisation of a reagent for generating free thiol from thioether groups and a related photocleavable, heterobifunctional crosslinking reagent. Chemical and spectroscopic studies of a model thiol protected derivative were used to show some features of thiol group photodeprotection. To demonstrate how the photocleavable crosslinking reagent may be used to modulate the activity of proteins we investigated the effect of light on the nucleating activity of crosslinked actin dimer; thus following near-ultraviolet irradiation of the actin dimer the crosslink was cleaved, presumeably at the thioether bond, resulting in the concomitant dissociation of dimer, loss of nucleating activity and creation of a concentration jump of polymerisable G-actin monomer. On the basis of this initial study we discuss applications and limitations of these reagents for the photomodulation of protein activity in vitro and in vivo.
Marriott
G
G
Department of Physics, Faculty of Science and Technology, Keio University, Yokohama, Japan.
Miyata
H
H
Kinosita
K
K
D000199:Actins / Q000737:chemistry / Q000528:radiation effects*; D003432:Cross-Linking Reagents; D066298:In Vitro Techniques; D009579:Nitrobenzoates; D010777:Photochemistry; D011487:Protein Conformation / Q000528:radiation effects; D013388:Succinimides; D013439:Sulfhydryl Reagents; D014466:Ultraviolet Rays
D000199:Actins; D003432:Cross-Linking Reagents; D009579:Nitrobenzoates; D013388:Succinimides; D013439:Sulfhydryl Reagents; C075154:4-bromomethyl-3-nitrobenzoic acid succinimide ester; C075153:4-bromomethyl-3-nitrobenzoic acid