PARATHYROID HORMONE

甲状旁腺激素（PTH）一直是许多研究的对象 多年来，由于其在监管方面的关键作用， 细胞外钙稳态 最近，越来越多的人 PTH和PTH衍生激动剂作为骨重建的合成代谢剂 并且作为治疗骨质疏松症的可能的候选药物， 被目击。 根据之前的结构调查，阿尔法 已经鉴定了N-和C-末端结构域中的螺旋。 从 序列截短研究表明，C-末端 重要的是结合到膜嵌入G蛋白偶联 受体的 鉴于这种螺旋的两亲性，我们提出 这个螺旋的作用是与膜表面相互作用， 作为锚，而PTH肽的N末端螺旋 激活受体。 为了检验这个假设，我们开始了 研究其中氨基酸22-31已被 被序列E（22）-L-L-E-K-L-L-E-KL（31）取代，设计为 模拟一个完美的两亲性α-螺旋，几乎没有序列同源性 在该区域（除两亲性之外）与天然PTH 激素. 如果结合结构域中的两亲性α-螺旋是 基序被受体识别，那么修饰应该产生 一种有效的激动剂 当然，这一结论的前提是， 修饰在肽序列内是螺旋状的。 解决 此时，开始对该肽进行NMR研究。 到 模拟研究肽的膜环境 DPC胶束的存在。 在这些条件下， 的质子信号被显著地加宽， 更差的光谱分辨率。 正因为如此， 把质子信号分配给室内的仪器 在750 MHz光谱仪上收集的质子-质子光谱允许 用于所有质子信号的明确分配， 确定了大量以前未解决的NOE。 的 化学位移指数和NOE清楚地表明， 螺旋的C-末端，并允许发展的高 这一类比的解析结构。

Parathyroid hormone (PTH) has been the object of numerous studies for many years now, owing to its crucial role in the regulation of extracellular calcium homeostasis. More recently, a growing interest in PTH and PTH-derived agonists as anabolic agents for bone remodeling and as possible drug candidates for the treatment of osteoporosis has been witnessed. From previous structural investigations, alpha helices in the N-and C-terminal domains have been identified. From sequence truncation studies, the C-terminus has been shown to be important for binding to the membrane embedded G-protein coupled receptor. Given the amphipathic nature of this helix, we proposed that the role of this helix is to interact with the membrane surface, to act as an anchor while the N-terminal helix of the PTH peptide activates the receptor. To examine this hypothesis, we began the investigation of an analog in which the amino acids 22-31 have been substituted by the sequence E(22)-L-L-E-K-L-L-E-KL(3 1), designed to model a perfect amphiphilic alpha-helix, with little sequence homology in this region (other than the amphiphilic nature) to the native PTH hormone. If an amphiphilic alpha-helix in the binding domain is the motif recognized by the receptor, then the modification should produce an effective agonist. Of course, this conclusion presupposes that the modification will be helical within the peptide sequence. To address this point, an NMR investigation of this peptide was initiated. To mimic the environment of the membrane the peptide was investigated in the presence of DPC micelles. Under these conditions the line widths of the proton signals are significantly broadened resulting in even worse spectral resolution. Because of this, it was impossible to even assign the proton signals with the instrumentation in house. Proton-proton spectra collected on the 750 MHz spectrometer allowed for the unambiguous assignment of all of the proton signals and identification of a large number of NOEs, previously unresolved. The chemical shift index and NOEs clearly indicated the presence of a helix in the C-terminus and allowed the development of a high resolution structure of this analog.