Trieu Melissa M, Devine Erin L, Lamarche Lindsey B, Ammerman Aaron E, Greco Jordan A, Birge Robert R, Theobald Douglas L, Oprian Daniel D
From the Department of Biochemistry, Brandeis University, Waltham, Massachusetts 02454 and.
the Departments of Chemistry and Molecular and Cell Biology, University of Connecticut, Storrs, Connecticut 06269.
J Biol Chem. 2017 Jun 23;292(25):10379-10389. doi: 10.1074/jbc.M117.789636. Epub 2017 May 4.
RhoGC is a rhodopsin (Rho)-guanylyl cyclase (GC) gene fusion molecule that is central to zoospore phototaxis in the aquatic fungus It has generated considerable excitement because of its demonstrated potential as a tool for optogenetic manipulation of cell-signaling pathways involving cyclic nucleotides. However, a reliable method for expressing and purifying RhoGC is currently lacking. We present here an expression and purification system for isolation of the full-length RhoGC protein expressed in HEK293 cells in detergent solution. The protein exhibits robust light-dependent guanylyl cyclase activity, whereas a truncated form lacking the 17- to 20-kDa N-terminal domain is completely inactive under identical conditions. Moreover, we designed several RhoGC mutants to increase the utility of the protein for optogenetic studies. The first class we generated has altered absorption spectra designed for selective activation by different wavelengths of light. Two mutants were created with blue-shifted (E254D, λ = 390 nm; D380N, λ = 506 nm) and one with red-shifted (D380E, λ = 533 nm) absorption maxima relative to the wild-type protein (λ = 527 nm). We also engineered a double mutant, E497K/C566D, that changes the enzyme to a specific, light-stimulated adenylyl cyclase that catalyzes the formation of cAMP from ATP. We anticipate that this expression/purification system and these RhoGC mutants will facilitate mechanistic and structural exploration of this important enzyme.
RhoGC是一种视紫红质（Rho）-鸟苷酸环化酶（GC）基因融合分子，是水生真菌游动孢子光趋性的核心。由于其作为一种用于光遗传学操纵涉及环核苷酸的细胞信号通路的工具所展现出的潜力，它引发了极大的关注。然而，目前缺乏一种可靠的表达和纯化RhoGC的方法。我们在此展示一种表达和纯化系统，用于在去污剂溶液中分离在HEK293细胞中表达的全长RhoGC蛋白。该蛋白表现出强大的光依赖性鸟苷酸环化酶活性，而缺少17至20 kDa N端结构域的截短形式在相同条件下完全无活性。此外，我们设计了几个RhoGC突变体，以增加该蛋白在光遗传学研究中的实用性。我们构建的第一类突变体具有改变的吸收光谱，旨在通过不同波长的光进行选择性激活。相对于野生型蛋白（λ = 527 nm），创建了两个蓝移突变体（E254D，λ = 390 nm；D380N，λ = 506 nm）和一个红移突变体（D380E，λ = 533 nm）。我们还构建了一个双突变体E497K/C566D，它将该酶转变为一种特异性的、光刺激的腺苷酸环化酶，可催化由ATP形成cAMP。我们预计这种表达/纯化系统和这些RhoGC突变体将有助于对这种重要酶进行机制和结构探索。
