Merzlyakov Mikhail, Chen Lirong, Hristova Kalina
Department of Materials Science and Engineering, Johns Hopkins University, Baltimore, MD 21218, USA.
J Membr Biol. 2007 Feb;215(2-3):93-103. doi: 10.1007/s00232-007-9009-0. Epub 2007 Jun 14.
The energetics of transmembrane (TM) helix dimerization in membranes and the thermodynamic principles behind receptor tyrosine kinase (RTK) TM domain interactions during signal transduction can be studied using Förster resonance energy transfer (FRET). For instance, FRET studies have yielded the stabilities of wild-type fibroblast growth factor receptor 3 (FGFR3) TM domains and two FGFR3 pathogenic mutants, Ala391Glu and Gly380Arg, in the native bilayer environment. To further our understanding of the molecular mechanisms of deregulated FGFR3 signaling underlying different pathologies, we determined the effect of the Gly382Asp FGFR3 mutation, identified in a multiple myeloma cell line, on the energetics of FGFR3 TM domain dimerization. We measured dimerization energetics using a novel FRET acquisition and processing method, termed "emission-excitation FRET (EmEx-FRET)," which improves the precision of thermodynamic measurements of TM helix association. The EmEx-FRET method, verified here by analyzing previously published data for wild-type FGFR3 TM domain, should have broad utility in studies of protein interactions, particularly in cases when the concentrations of fluorophore-tagged molecules cannot be controlled.
利用荧光共振能量转移（FRET），可以研究膜中跨膜（TM）螺旋二聚化的能量学以及信号转导过程中受体酪氨酸激酶（RTK）TM结构域相互作用背后的热力学原理。例如，FRET研究得出了野生型成纤维细胞生长因子受体3（FGFR3）TM结构域以及两种FGFR3致病突变体Ala391Glu和Gly380Arg在天然双层膜环境中的稳定性。为了进一步了解不同病理状态下FGFR3信号失调的分子机制，我们确定了在多发性骨髓瘤细胞系中鉴定出的Gly382Asp FGFR3突变对FGFR3 TM结构域二聚化能量学的影响。我们使用一种名为“发射-激发FRET（EmEx-FRET）”的新型FRET采集和处理方法来测量二聚化能量学，该方法提高了TM螺旋缔合热力学测量的精度。通过分析先前发表的野生型FGFR3 TM结构域数据验证的EmEx-FRET方法，在蛋白质相互作用研究中应具有广泛的用途，特别是在无法控制荧光团标记分子浓度的情况下。
