Kopra Kari, Ligabue Alessio, Wang Qi, Syrjänpää Markku, Blaževitš Olga, Veltel Stefan, van Adrichem Arjan J, Hänninen Pekka, Abankwa Daniel, Härmä Harri
Institute of Biomedicine, Department of Cell Biology and Anatomy, Laboratory of Biophysics, University of Turku, Tykistökatu 6A 5th floor, 20520, Turku, Finland,
Anal Bioanal Chem. 2014 Jul;406(17):4147-56. doi: 10.1007/s00216-014-7795-7. Epub 2014 Apr 24.
A quenching resonance energy transfer (QRET) assay for small GTPase nucleotide exchange kinetic monitoring is demonstrated using nanomolar protein concentrations. Small GTPases are central signaling proteins in all eukaryotic cells acting as a "molecular switches" that are active in the GTP-state and inactive in the GDP-state. GTP-loading is highly regulated by guanine nucleotide exchange factors (GEFs). In several diseases, most prominently cancer, this process in misregulated. The kinetics of the nucleotide exchange reaction reports on the enzymatic activity of the GEF reaction system and is, therefore, of special interest. We determined the nucleotide exchange kinetics using europium-labeled GTP (Eu-GTP) in the QRET assay for small GTPases. After GEF catalyzed GTP-loading of a GTPase, a high time-resolved luminescence signal was found to be associated with GTPase bound Eu-GTP, whereas the non-bound Eu-GTP fraction was quenched by soluble quencher. The association kinetics of the Eu-GTP was measured after GEF addition, whereas the dissociation kinetics could be determined after addition of unlabeled GTP. The resulting association and dissociation rates were in agreement with previously published values for H-Ras(Wt), H-Ras(Q61G), and K-Ras(Wt), respectively. The broader applicability of the QRET assay for small GTPases was demonstrated by determining the kinetics of the Ect2 catalyzed RhoA(Wt) GTP-loading. The QRET assay allows the use of nanomolar protein concentrations, as more than 3-fold signal-to-background ratio was achieved with 50 nM GTPase and GEF proteins. Thus, small GTPase exchange kinetics can be efficiently determined in a HTS compatible 384-well plate format.
一种用于监测小GTP酶核苷酸交换动力学的猝灭共振能量转移（QRET）检测方法，可在纳摩尔蛋白浓度下进行。小GTP酶是所有真核细胞中的核心信号蛋白，充当“分子开关”，在GTP状态下活跃，在GDP状态下不活跃。GTP加载受到鸟嘌呤核苷酸交换因子（GEF）的高度调节。在几种疾病中，最显著的是癌症，这个过程失调。核苷酸交换反应的动力学反映了GEF反应系统的酶活性，因此特别受关注。我们在小GTP酶的QRET检测中使用铕标记的GTP（Eu-GTP）来测定核苷酸交换动力学。在GEF催化GTP酶的GTP加载后，发现与GTP酶结合的Eu-GTP相关的高时间分辨发光信号，而未结合的Eu-GTP部分被可溶性猝灭剂猝灭。在加入GEF后测量Eu-GTP的结合动力学，而在加入未标记的GTP后可以确定解离动力学。所得的结合和解离速率分别与先前发表的H-Ras（Wt）、H-Ras（Q61G）和K-Ras（Wt）的值一致。通过测定Ect2催化的RhoA（Wt）GTP加载的动力学，证明了QRET检测对小GTP酶具有更广泛的适用性。QRET检测允许使用纳摩尔蛋白浓度，因为在50 nM GTP酶和GEF蛋白的情况下实现了超过3倍的信噪比。因此，可以在适用于高通量筛选的384孔板形式中有效地测定小GTP酶的交换动力学。
