Conformational Dynamics of Ligand Binding Domains of GluR2

离子型谷氨酸受体（iGluR）是介导中枢神经系统兴奋性信号传导的配体门控离子通道。 在配体与iGluR的胞外结构域结合后，随后发生局部构象变化，并且这种运动被翻译为跨膜结构域，从而诱导通道开放。 我们使用了一个分离的配体结合域（LBD），GluR 2-S1 S2 J（GluR 2），作为一个模型系统来研究蛋白质-配体复合物。使用时间分辨荧光和各向异性测量，我们的特点是激发态的性质和本地流动性的色氨酸残基在孤立的LBD，GluR 2。 具体而言，我们确定了广泛使用的和结构特征的拮抗剂，6，7-二硝基喹喔啉-2，3-二酮（DNQX）作为一个有效的荧光能量转移（FET）受体色氨酸。 与晶体学数据相一致，我们的结果表明，四个原生的Dahans是福斯特半径（33埃）内的结合配体。 此外，我们通过鉴定GluR 2的原始FET配体3-硝基酪氨酸（3 NY）（24埃，表观解离常数，Kd约为170微摩尔）证明了该技术的更广泛价值。 从色氨酸激发态衰变中提取的估计平均供体-受体（色氨酸-配体）距离对于两种配体（24埃）是相似的，这表明3 NY结合在结构上表征的配体结合裂缝中。 有趣的是，我们观察到3 NY的多个速率分量，提示配体-蛋白质复合物的结构异质性。 然而，由于存在多个Trp供体，我们只能估计到Trp残基的平均DNQX/3 NY距离。 虽然从我们对DNQX-Trp GluR 2的分析中提取的距离（24埃）与晶体学确定的距离（12-20埃）相当，但它们略长。 这可能是由于当前可分辨速率的限制（τ为0.5 ns），其对应于比Trp-DNQX估计的供体-受体距离短的供体-受体距离，或者可能归因于折叠蛋白中Trp和DNQX过渡偶极子的不利取向。 或者，我们更长的距离可能只是反映了蛋白质在溶液中的动力学性质。 虽然DNQX是一种众所周知的复合物，但其光谱特性和作为配体与GluR 2结合的敏感报告分子的潜在应用在以前被忽视了。 我们已经描述了DNQX竞争测定，其代表了对目前使用的荧光测定的显著改进，因为这是一种开启传感器，消除了由光漂白引起的淬灭事件所固有的假阳性观察。 使用FET竞争配体和固有的Trp荧光应该允许未来的新的配体的鉴定，不仅为GluR 2，但也为其他蛋白质时，DNQX或结构类似物（即CNQX和其他硝化芳香族化合物）被采用。

Ionotropic glutamate receptors (iGluRs) are ligand-gated ion channels that mediate excitatory signaling in the central nervous system. Upon ligand binding to the extracellular domain of iGluRs local conformational changes ensue and this motion is translated to the transmembrane domain inducing channel opening. We have used an isolated ligand binding domain (LBD), GluR2-S1S2J (GluR2), as a model system to study the protein-ligand complex. Using time-resolved fluorescence and anisotropy measurements, we characterized the excited state properties and local mobility of Trp residues in the isolated LBD, GluR2. Specifically, we determined that the widely used and structurally characterized antagonist, 6,7-dinitroquinoxaline-2,3-dione (DNQX) acts as an efficient fluorescence energy transfer (FET) acceptor for Trp. Consistent with crystallographic data, our results indicate that the four native tryptophans are within Forsters radius (33 angstroms ) of the bound ligand. Additionally, we demonstrate the broader value of this technique by identifying an original FET ligand, 3-nitrotyrosine (3NY) for GluR2 (24 angstroms , apparent dissociation constant, Kd of approximately 170 micromolar). Estimated average donor-acceptor (Trp-to-ligand) distances extracted from tryptophan excited-state decays are similar for both ligands (24 angstroms) suggesting that 3NY binds in the structurally characterized ligand-binding cleft. Interestingly, we observe multiple rate components for 3NY suggestive of ligand-protein complex structural heterogeneity. However, due to the presence of multiple Trp donors we are only able to estimate average DNQX/3NY distances to the Trp residues. While the distance extracted (24 angstroms) from our analysis on DNQX-Trp GluR2 are on the order of the crystallographically determined distances (12-20 angstroms), they are somewhat longer. This may be due to the current limit of resolvable rates (tau of 0.5 ns) which would correspond to donor-acceptor distances shorter than those estimated for Trp-DNQX or it is possible that it is attributable to unfavorable orientations of the Trp and DNQX transition dipoles in the folded protein. Alternatively, our longer distances may simply reflect dynamical properties of the protein in solution. While DNQX is a well-known complex, its spectroscopic properties and potential application as a sensitive reporter for ligand binding to GluR2 have been overlooked previously. We have described a DNQX competition assay that represents a significant improvement over the fluorescence assays currently in use in that this is a turn-on sensor, eliminating false positive observations inherent to quenching events resulting from photobleaching. The use of FET competitor ligands and intrinsic Trp fluorescence should allow for the future identification of novel ligands for not only for GluR2 but also for other proteins when DNQX or structural analogs (i.e. CNQX and other nitrated aromatic compounds) are employed.