Design and characterization of an intramolecular antiparallel coiled coil peptide.
Design and characterization of an intramolecular antiparallel coiled coil peptide.
复制标题
分子内反平行卷曲螺旋肽的设计和表征。
DOI：
10.1021/bi00175a003
复制
发表时间：
1994
期刊：
影响因子：
2.9
通讯作者：
Chaiken,IM
中科院分区：
文献类型：
--
作者：
Myszka,DG;Chaiken,IM
19938年12月6日收到的修订版摘要：一种56个残基的多肽被设计成一个稳定的分子内反平行卷曲螺旋，称为卷曲螺旋茎环。α-螺旋的反平行取向由七肽重复序列（a，B，c，d，e，f，g）n中疏水和离子残基的比对决定。卷曲螺旋界面处的疏水核心分别被七肽位置d anda'和位置a和d'中的亮氨酸和缬氨酸残基占据。界面边界位置e和g被氨基末端螺旋中的谷氨酸和羧基末端螺旋中的赖氨酸残基占据。连接α-螺旋的环段以螺旋断裂残基甘氨酸和脯氨酸开始和结束。丙氨酸和丝氨酸残基被放置在两个螺旋的暴露的db、c和f位置上，以分别增加肽的螺旋倾向和溶解度。几条证据表明，用这种设计制备的合成肽折叠成稳定的单体卷曲螺旋茎环构象：（1）该肽在pH 7.4的150 mM氯化钠和50 mM磷酸钠中高度可溶;（2）圆二色光谱是α-螺旋的，但在222和208 nm处具有卷曲螺旋结构特征的相对椭圆率最小值;（3）该肽的α-螺旋含量接近80%，与肽浓度无关，在三氟乙醇存在下无变化，（4）体积排阻色谱和沉降平衡超滤测定证实该肽在水溶液中为单体;（5）该肽在较宽的pH范围内显示出高的螺旋含量，（6）展开α-螺旋结构的表观Tm大于65 ℃，并且需要3.0M尿素来将螺旋结构减少50%;（7）在单体的氨基端和羧基端半胱氨酸残基之间容易形成二硫键，证实了螺旋的反平行取向;和（8）肽与纤维蛋白原竞争GPIIbllla受体，表明存在于环序列中的RGD残基可用于结合。这项工作确立了α-螺旋的反平行排列可以通过设计卷曲螺旋内的特定疏水和离子相互作用来实现。卷曲螺旋肽是一种序列简化的支架，可以将天然蛋白质的α-螺旋和环插入其中，形成构象受限的模拟识别分子，折叠大分子的识别是一个反复出现的主题，基本上是所有生物过程的基础。模拟大分子的结合位点可以提供在生物技术中用于治疗、诊断和分离的识别分子。生物大分子如蛋白质可以被认为是构象框架或支架的复合物，其中有限数量的识别元件存在于表面上。因此，从头构建理想化的支架，其中嵌入来自天然存在的蛋白质和肽的识别元件，可以提供模拟物和拮抗剂设计的通用方法。
Revised Manuscript Received December 6, 19938 abstract: A 56-residue polypeptide was designed to fold into a stable intramolecular antiparallel coiled coil, referred to as a coiled coil stem loop. The antiparallel orientation of the a-helices was dictated by the alignment of hydrophobic and ionic residues in the heptad repeat sequence (a, b, c, d, e, f, g)„. The hydrophobic core at the coiled coil interface was occupied by leucine and valine residues in heptad positions d anda'and positions a and d', respectively. The interface border positions e and g were occupied by glutamic acid in the amino-terminal helix and lysine residues in the carboxy-terminal helix. A loop segment connecting the a-helices began and ended with the helix-breaking residues glycine and proline. Alanine and serine residues were placed on the exposedb, c, and f positions of both helices to increase the helical propensity and solubility of the peptide, respectively. Several lines of evidence argued that the synthetic peptide made with this design folded into a stable monomeric coiled coil stem loop conformation:(1) the peptide was highly soluble in 150 mM sodium chloride and 50 mM sodium phosphate, pH 7.4;(2) the circular dichroism spectrum was a-helical but with relative ellipticity minima at 222 and 208 nm characteristic of a coiled coil structure;(3) the peptide exhibited an a-helical content near 80%, which was independent of peptide concentration and unchanged in the presence of trifluoroethanol;(4) size exclusion chromatography and sedimentation equilibrium ultracentrifuge measurements confirmed that the peptide was monomeric in aqueous solution;(5) the peptide exhibited high helical content over a wide pH range;(6) the apparent Tm for unfolding the a-helical structure was greater than 65 C, and 3.0 M urea was required to reduce the helical structure by 50%;(7) a disulfide bond was readily formed in the monomer between the aminoand carboxy-terminal cysteine residues, confirming the antiparallel orientation of the helices; and (8) the peptide competed with fibrinogen for the GPIIbllla receptor indicating that the RGD residues present in the loop sequence were available for binding. This work establishes that an antiparallel alignment of a-helices can be achieved by designing specific hydrophobic and ionic interactions within the coiled coil. The prototype coiled coil peptide represents a sequence-simplified scaffold into which residues from a-helices and loops of native proteins can be inserted to form conformationally constrained mimetic recognition molecules.Recognition of folded macromolecules is a recurrent theme which underlies essentially all biological processes. Mimicking binding sites of macromolecules can provide recognition molecules of use in biotechnology, for therapeutics, diagnostics, and separation. Biological macromolecules such as proteins can be thought of as composites of conformational frameworks, or scaffolds, in which a limited number of recognition elements are presented on the surface. Hence, de novo construction of idealized scaffolds, in which recognition elements from naturally occurring proteins and peptides are embedded, can provide a generalized approach to mimetics and antagonist design.
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影响因子：
2.9
作者：
GRADDIS, TJ;MYSZKA, DG;CHAIKEN, IM
通讯作者：
CHAIKEN, IM
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DOI：
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DOI：
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发表时间：
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期刊：
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作者：
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作者：
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DOI：
--
发表时间：
1984
期刊：
影响因子：
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作者：
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通讯作者：
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