HELIX-TURN-HELIX PEPTIDES THAT FORM A-HELICAL FIBRILS

这个子项目是利用由NIH/NCRR资助的中心拨款提供的资源的许多研究子项目之一。子项目和调查员(PI)可能从另一个NIH来源获得了主要资金，因此可能会出现在其他CRISE条目中。列出的机构是针对中心的，而不一定是针对调查员的机构。高密度脂蛋白(HDL)大小的主要决定因素是其主要蛋白载脂蛋白A-I(ApoA-I)。载脂蛋白A-I的模型预测，它包含10个交替连接的两亲性α-螺旋片段。我们合成了四个带有两个18个残基的两亲性α-螺旋片段的多肽(Anantharamaiah，G.M.等人，(1985)J.Biol)。化学。10248-10255)，由来自载脂蛋白A-I的假定的转弯序列连接。1.Ac-DWLKAFYDKVAEKLKEAFKVEPLRADWLKAFYDKVAEKLKEAF-NH2 2.Ac-DWLKAFYDKVAEKLKEAFGLLPVLEDWLKAFYDKVAEKLKEAF-NH2 3.Ac-DWLKAFYDKVAEKLKEAFKVQPYLDDWLKAFYDKVAEKLKEAF-NH2 4.Ac-DWLKAFYDKVAEKLKEAFNGGARLADWLKAFYDKVAEKLKEAF-NH2多肽1-4在37℃、10 mM磷酸二氢钠、pH 7.60中孵育后形成沉淀物。然而，与典型的β-折叠淀粉样纤维的预期相反，肽1和2形成的纤维不与硫代黄素T结合，并导致刚果红光谱发生蓝移。用圆二色谱(CD)和傅里叶变换红外光谱(FTIR)对多肽1和2的原纤维进行了表征，发现它们具有明显的α螺旋特征。在磁性排列为1的样品上进行的同步辐射x射线光纤衍射证实了原纤维中的α螺旋特征。这些数据表明，由肽1-4形成的沉淀物或纤维在结构上是不同的。多肽1和2形成螺旋状原纤维，螺旋轴垂直于主原纤轴。与1和2相比，3和4在37℃孵育30天后仅观察到少量沉淀，这是硫代黄素T和刚果红结合的典型淀粉样纤维。这些研究表明，较长的两亲性α-螺旋片段之间不同的旋转序列可以调节纤维的结构。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. A major determinant of High Density Lipoprotein (HDL) size is its main protein, Apolipoprotein A-I (apo A-I). Models of apo A-I predict that it contains 10 amphiphilic alpha-helical segments connected by turns. We synthesized four peptides with two 18-residue, amphiphilic alpha-helical segments (Anantharamaiah, G. M. et al., (1985) J. Biol. Chem. 260, 10248-10255) connected by putative turn sequences from apo A-I. 1. Ac-DWLKAFYDKVAEKLKEAFKVEPLRADWLKAFYDKVAEKLKEAF-NH2 2. Ac-DWLKAFYDKVAEKLKEAFGLLPVLEDWLKAFYDKVAEKLKEAF-NH2 3. Ac-DWLKAFYDKVAEKLKEAFKVQPYLDDWLKAFYDKVAEKLKEAF-NH2 4. Ac-DWLKAFYDKVAEKLKEAFNGGARLADWLKAFYDKVAEKLKEAF-NH2 Peptides 1-4 formed precipitates after incubating them at 37¿C in 10 mM sodium phosphate, pH 7.60. In contrast to what is expected for typical beta-sheet amyloid fibrils, however, peptides 1 and 2 formed fibrils that did not bind thioflavin T and induced a blue shift in the spectrum of Congo red. Circular dichroism (CD) and Fourier Transform Infrared Spectroscopy (FTIR) of the fibrils from peptides 1 and 2 show significant alpha-helical character. Synchrotron x-ray fiber diffraction on a magnetically aligned sample of 1 has confirmed the alpha-helical character in the fibrils. These data indicate that precipitates or fibrils formed from peptides 1-4 are structurally diverse. Peptides 1 and 2 formed ?-helical fibrils, with helical axes perpendicular to the main fibril axis. In contrast to 1 and 2, only a small amount of precipitate was observed after incubating 3 and 4 at 37¿C for 30 days, and these are typical amyloid fibrils by thioflavin T and Congo red binding. These studies indicate that varying turn sequences between longer amphiphilic alpha-helical segments can modulate the structure of fibrils.