Determining stoichiometry in homomultimeric nucleic acid complexes using magnetic field induced residual dipolar couplings
Determining stoichiometry in homomultimeric nucleic acid complexes using magnetic field induced residual dipolar couplings
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DOI：
10.1021/ja0105865
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发表时间：
2001-06-20
影响因子：
15
通讯作者：
Patel, DJ
中科院分区：
文献类型：
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作者：
Al-Hashimi, HM;Tolman, JR;Patel, DJ
核酸寡聚化是驱动参与各种识别事件的结构元件形成的重要步骤。高阶DNA结构被认为是端粒、着丝粒和三联体疾病序列元件中的识别基序，1而逆转录病毒RNA同源二聚化为“双链体”和“接吻”复合物在病毒复制和基因组包装的各个阶段中起着重要作用。2尽管寡聚化很重要，但由于对称同源多聚体中出现的化学位移简并性，传统上很难通过核磁共振（NMR）光谱3直接表征核酸多聚体状态。除了剥夺洞察力的热力学因素，支配核酸在溶液中的寡聚化，这种简并性可以阻碍多聚化学计量的测定，4这是至关重要的准确解释NMR距离的限制在高分辨率结构测定。5，6在这里，我们介绍了一种新的方法，用于探测核酸分子间的相互作用，依赖于磁场诱导的残余偶极耦合（fiRDC）的测量，7并证明了应用程序的直接测定多聚体化学计量在高阶DNA架构。在高磁场（B）下，核酸分子呈现足够水平的分子对齐以允许测量作为对正常观察到的标量偶联的贡献的fiRDC。7观察到的fiRDC的幅度二次地取决于磁场强度和磁化率张量的主值（ii i）{x，y，z}），其对于核酸由芳族碱基的抗磁磁化率（ii（base））支配。7 e由于堆积相互作用有利于核酸中碱基平面的共面排列，因此它们对应的张量往往接近轴对称（yy xx），6，8主值（ii）由于碱基亲和性的建设性增加而随着碱基总数近似线性增加（图1）。因此，对多聚体（（m-mer））测定的实验ii值与对单体（（1-mer））预期的相应值之间的比较可以提供用于测定多聚体化学计量的新途径。在下文中，我们开发了一个独立于先验结构信息的确定框架。一般来说，主值ii（m聚体）可以从RDC值Dii推导出，该值Dii是针对沿着相应的第i个主方向取向的相互作用矢量测量的。9对于核酸，yy是一个很好的实验测定目标。首先，由于碱基相互作用矢量垂直于其自身的主各向异性（zz（碱基）），因此它们将优先位于总（m-mer）主轴系统的yy-xx平面内（图1）。第二，由于核酸倾向于具有接近轴向的双张量，6，8在yy-xx平面中为相互作用矢量测量的许多RDC值将提供对Dyy值的良好估计。此外，由于从扩展的抗磁性核酸（zz< 0）预期的负磁化率各向异性，Dyy的值将对应于来自给定测量组的最大RDC值（D+）（对于1DNH为+ ve，对于1DCH为-ve）。因此，D+的观察值提供了Dyy值的估计，其又与所需的总多聚体yy（m-mer）值直接对应，其中所有符号具有其通常的含义。虽然原则上任何相互作用载体都可以用于确定D+，但我们将重点关注直接相互作用载体之间的fiRDC。
Nucleic acid oligomerization is an important step for driving formation of structural elements that are involved in a variety of recognition events. Higher-order DNA architectures are implicated as recognition motifs in elements of telomeric, centromeric, and triplet disease sequences, 1 while homodimerization of retroviral RNA into “duplex” and “kissing” complexes plays an important role in various stages of viral replication and genome packaging. 2 Despite the importance of oligomerization, direct characterization of nucleic acid multimeric states by nuclear magnetic resonance (NMR) spectroscopy3 has traditionally been difficult owing to the chemical shift degeneracy that arises in symmetric homomultimers. In addition to depriving insight into thermodynamic factors that govern nucleic acid oligomerization in solution, this degeneracy can hinder determination of multimeric stoichiometry, 4 which is of critical importance for accurate interpretation of NMR distance constraints during high-resolution structure determination. 5, 6 Here, we introduce a new approach for probing intermolecular interactions in nucleic acids that relies on the measurement of magnetic field induced residual dipolar couplings (fiRDCs), 7 and demonstrate an application to the direct determination of multimeric stoichiometry in higher-order DNA architectures. Under high magnetic fields (B), nucleic acid molecules assume a sufficient level of molecular alignment to allow measurement of fiRDCs as contributions to normally observed scalar couplings. 7 The magnitudes of observed fiRDCs depend quadratically on the magnetic field strength, and on the principal values of the magnetic susceptibility tensor (ii i){x, y, z}), which for nucleic acids are dominated by the diamagnetic susceptibilities of aromatic base groups (ii(base)). 7e Because stacking interactions favor coplanar arrangement of base planes in nucleic acids, their corresponding-tensors tend to be close to axially symmetric (yy≈ xx), 6, 8 with principal values (ii) that increase approximately linearly with the total number of bases due to constructive addition of base susceptibilities (Figure 1). Thus, comparisons between experimental ii values determined for a multimer ((m-mer)) with corresponding values expected for a monomer ((1-mer)) can provide a new route for the determination of multimeric stoichiometry. In what follows, we develop a framework for this determination that is independent of a priori structural information. In general, a principal value, ii(m-mer), can be derived from the RDC value, Dii, measured for an interaction vector oriented along the corresponding ith principal direction. 9 For nucleic acids, yy is a good target for experimental determination. First, because base interaction vectors are perpendicular to their own principal anisotropy (zz (base)), they will preferentially be positioned within the yy-xx plane of the total (m-mer) principal axis system (Figure 1). Second, as nucleic acids tend to have close to axially symmetric-tensors, 6, 8 many RDC values measured for interaction vectors in the yy-xx plane will provide a good estimate for the value of Dyy. Moreover, due to the negative susceptibility anisotropy expected from extended diamagnetic nucleic acids (zz< 0), the value of Dyy will correspond to the largest RDC value (D+)(+ ve for 1DNH and-ve for 1DCH) from a given set of measurements. The observed value for D+ hence provides an estimate for the value of Dyy, which in turn has a direct correspondence with the desired total multimeric yy(m-mer) value, where all symbols have their usual meaning. While in principle any interaction vector can be used to determine D+, we will focus on fiRDCs between directly …