Elucidation of Structural Effect of C-terminal Amidation of Bioactive Peptide

1) c端修饰和非修饰二肽的x射线晶体结构分析为了阐明c端α-酰胺化引起的多肽的一系列结构特征，采用x射线衍射方法分析了H-Val-Gly-NH2、h - ser - ph - nh2、H-Gly-Tyr-NH2和H-Pro-Tyr-NH2盐酸盐的晶体结构。虽然各自的分子在主链和侧链上都具有能量允许的扭转角，但它们的构象不一定与相应的未经修饰的构象相同。这是由于不同的分子包装要求，而不是不同的构象特征固有的c -修饰和-未修饰肽。在分子填充特征上，每个肽都倾向于通过酰胺NH和O=C基团共同参与的氢键形成重复结构。氯离子位于相邻肽之间，并与各自的酰胺NHs形成氢键，从而导致更多的薄片结构。在分析结果的基础上，讨论了酰胺基团的氢键特性及其在分子填充中的作用。为了明确内源性m受体配体内源性m受体配体内源性m受体配体内源性m受体配体内源性m受体配体内源性m受体配体内源性m受体配体内源性m受体2的c端酰胺基团（EM2, h - tyrr - pro - pheh - pheh - nh2）在生物功能中的结构作用，通过二维^1H-NMR测量和分子模拟计算，比较了内源性m受体2及其c端游离酸（EM2OH, h - tyrr - pro - pheh - pheh -OH）的溶液构象。这两种多肽在TFE和水中以1:2的比例分布在tir - pro - w键周围的顺式和反式异构体之间，仅在渗透氘化十二烷基磷酸胆碱（DPC）的膜模拟胶束中处于反式平衡。利用动力学模拟退火方法，在由ROE交叉峰推导出的质子-质子距离约束下，生成了每种异构体的50种可能的三维结构。EM2构象在TFE和水中均为开放构象，而EM2OH在伸展和折叠主结构之间表现出构象变化。另一方面，DPC中的EM2以2:1的比例呈现伸展和折叠的主链结构，而EM2OH则呈现开放的构象。这些结果清楚地表明羧基取代c端酰胺基使肽具有柔韧性。通过对EM2和EM2OH的构象特征的比较，讨论了μ受体激活的构象要求。少

1)X-ray crystal structure analyses of C-terminal amidated and nonamidated dipeptidesAs a series of elucidating the structural features of peptides caused by the C-terminal α-amidation, the crystal structures of H-Val-Gly-NH2, H-Ser-Phe-NH2, H-Gly-Tyr-NH2, and H-Pro-Tyr-NH2 hydrochloride salts were analyzed by X-ray diffraction method. Although respective molecules take the energetically allowable torsion angles concerning the backbone and side chains, their conformations are not necessarily the same as the corresponding unamidated ones. This is resulted from the different molecular packing requirement, rather than the different conformational feature inherent in the C-amidated and -unamidated peptides. As for the molecular packing feature, each peptide tends to form the repeated structure through the hydrogen bonds in which both amide NH and O=C groups participate. The chloride ions are located between the neighboring peptides and are hydrogen-bonded to the respective amide NHs, leadin … More g to the sheet structure. The hydrogen-bonding feature of amide group and its function for the molecular packing has been discussed based on the results so far analyzed.2)Analyses of solution structures of m-opioid agonist endomorphin 2 and its C-terminal OH formIn order to make clear the structural role of the C-terminal amide group of endomorphin-2 (EM2, H-Tyr-Pro-Phe-Phe-NH2), an endogenous m-receptor ligand, for the biological function, the solution conformations of endomorphin-2 and its C-terminal free acid (EM2OH, H-Tyr-Pro-Phe-Phe-OH), studied by two-dimensional ^1H-NMR measurements and molecular modeling calculations, were compared. Both peptides were in equilibrium between the cis and trans isomers around the Tyr-Pro w bond in a population of approximately 1:2 ratio in TFE and water and only trans in the membrane-mimetic micelles of perdeuterated dodecylphosphocholine (DPC). Fifty possible 3D structures for the each isomer were generated by the dynamical simulated annealing method under the proton-proton distance constraints derived from the ROE cross peaks. EM2 conformers adopt an open conformation in both TFE and water, whereas EM2OH shows conformational variation between the extended and folded backbone structures. On the other hand, EM2 in DPC takes the extended and folded backbone structures in 2:1 ratio, whereas EM2OH shows an open conformation. These results indicate clearly that the substitution of carboxyl group for C-terminal amide group makes the peptide flexible. The conformational requirement for μ-receptor activation has been discussed based on the active form proposed for endomorphin-1 and by comparing conformational features of EM2 and EM2OH. Less