Bravin Carlo, Duindam Nol, Hunter Christopher A
Yusuf Hamied Department of Chemistry, University of Cambridge Lensfield Road Cambridge CB2 1EW UK
Chem Sci. 2021 Oct 13;12(42):14059-14064. doi: 10.1039/d1sc04741h. eCollection 2021 Nov 3.
Reversible formation of covalent adducts between a thiol and a membrane-anchored Michael acceptor has been used to control the activation of a caged enzyme encapsulated inside vesicles. A peptide substrate and papain, caged as the mixed disulfide with methane thiol, were encapsulated inside vesicles, which contained Michael acceptors embedded in the lipid bilayer. In the absence of the Michael acceptor, addition of thiols to the external aqueous solution did not activate the enzyme to any significant extent. In the presence of the Michael acceptor, addition of benzyl thiol led to uncaging of the enzyme and hydrolysis of the peptide substrate to generate a fluorescence output signal. A charged thiol used as the input signal did not activate the enzyme. A Michael acceptor with a polar head group that cannot cross the lipid bilayer was just as effective at delivering benzyl thiol to the inner compartment of the vesicles as a non-polar Michael acceptor that can diffuse across the bilayer. The concentration dependence of the output signal suggests that the mechanism of signal transduction is based on increasing the local concentration of thiol present in the vesicles by the formation of Michael adducts. An interesting feature of this system is that enzyme activation is transient, which means that sequential addition of aliquots of thiol can be used to repeatedly generate an output signal.
硫醇与膜锚定的迈克尔受体之间可逆形成共价加合物，已被用于控制包裹在囊泡内的笼形酶的激活。一种肽底物和木瓜蛋白酶，与甲硫醇形成混合二硫键而被笼蔽，被包裹在囊泡内，囊泡中含有嵌入脂质双层的迈克尔受体。在没有迈克尔受体的情况下，向外部水溶液中添加硫醇不会在任何显著程度上激活酶。在有迈克尔受体的情况下，添加苄硫醇会导致酶的去笼蔽以及肽底物的水解，从而产生荧光输出信号。用作输入信号的带电荷硫醇不会激活酶。具有不能穿过脂质双层的极性头部基团的迈克尔受体，在将苄硫醇递送至囊泡内部隔室方面，与能够扩散穿过双层的非极性迈克尔受体一样有效。输出信号的浓度依赖性表明，信号转导机制是基于通过形成迈克尔加合物来增加囊泡中硫醇的局部浓度。该系统的一个有趣特征是酶激活是短暂的，这意味着逐份添加硫醇可用于反复产生输出信号。
