Isaac Binumol, Gallagher Greg J, Balazs Yael S, Thompson Lynmarie K
Department of Chemistry, University of Massachusetts, Amherst, Massachusetts 01003-9336, USA.
Biochemistry. 2002 Mar 5;41(9):3025-36. doi: 10.1021/bi015759h.
The serine receptor of bacterial chemotaxis is an ideal system in which to investigate the molecular mechanism of transmembrane signaling. Solid-state nuclear magnetic resonance (NMR) techniques such as rotational resonance provide a means for measuring local structure and ligand-induced structural changes in intact membrane proteins bound to native membrane vesicles. A general site-directed biosynthetic (13)C labeling strategy is used to direct the distance measurements to a specific site; the distance is measured between a unique Cys residue and a non-unique, low-abundance residue (Tyr or Phe). A (13)C-(13)C internuclear distance measurement from (13)CO(i) to (13)C beta(i + 3) at the periplasmic edge of the second membrane-spanning helix (TM2) of 5.1 +/- 0.2 A is consistent with the predicted alpha-helical structure and thus demonstrates an accurate long-distance rotational resonance measurement in the 120 kDa membrane-bound receptor. These measurements require a correction for the rotational resonance exchange between the multiple labels of the non-unique amino acid and the natural-abundance (13)C, which is critical to distance measurements in complex systems. A second (13)C-(13)C distance measurement between the transmembrane helices provides a high-resolution measurement of tertiary structure in the transmembrane region. The measured 5.0-5.3 A distance in the presence and absence of ligand is consistent with structural models for the transmembrane region and a proposed signaling mechanism in which ligand binding induces a 1.6 A translation of TM2. This approach can be used for additional measurements of the structure of the transmembrane region and to determine whether the ligand-induced motion is indeed propagated through the transmembrane helices.
细菌趋化性的丝氨酸受体是研究跨膜信号传导分子机制的理想系统。诸如旋转共振等固态核磁共振（NMR）技术提供了一种测量与天然膜囊泡结合的完整膜蛋白的局部结构和配体诱导的结构变化的方法。一种通用的定点生物合成（13）C标记策略用于将距离测量导向特定位点；测量的是一个独特的半胱氨酸残基与一个非独特的、低丰度残基（酪氨酸或苯丙氨酸）之间的距离。在第二个跨膜螺旋（TM2）的周质边缘，从（13）CO（i）到（13）Cβ（i + 3）的（13）C-（13）C核间距离测量值为5.1±0.2埃，与预测的α螺旋结构一致，因此证明了在120 kDa膜结合受体中进行的准确的长距离旋转共振测量。这些测量需要对非独特氨基酸的多个标记与天然丰度（13）C之间的旋转共振交换进行校正，这对于复杂系统中的距离测量至关重要。跨膜螺旋之间的第二次（13）C-（13）C距离测量提供了跨膜区域三级结构的高分辨率测量。在存在和不存在配体的情况下测量的5.0 - 5.3埃距离与跨膜区域的结构模型以及一种提出的信号传导机制一致，在该机制中配体结合诱导TM2平移1.6埃。这种方法可用于对跨膜区域结构的额外测量，并确定配体诱导的运动是否确实通过跨膜螺旋传播。
