Wang Weixun, Hecht Michael H
Department of Chemistry, Princeton University, Princeton, NJ 08544-1009, USA.
Proc Natl Acad Sci U S A. 2002 Mar 5;99(5):2760-5. doi: 10.1073/pnas.052706199.
Amyloid fibrils are associated with a variety of neurodegenerative maladies including Alzheimer's disease and the prion diseases. The structures of amyloid fibrils are composed of beta-strands oriented orthogonal to the fibril axis ("cross beta" structure). We previously reported the design and characterization of a combinatorial library of de novo beta-sheet proteins that self-assemble into fibrillar structures resembling amyloid. The libraries were designed by using a "binary code" strategy, in which the locations of polar and nonpolar residues are specified explicitly, but the identities of these residues are not specified and are varied combinatorially. The initial libraries were designed to encode proteins containing amphiphilic beta-strands separated by reverse turns. Each beta-strand was designed to be seven residues long, with polar (open circle) and nonpolar (shaded circle) amino acids arranged with an alternating periodicity ([see text]). The initial design specified the identical polar/nonpolar pattern for all of the beta-strands; no strand was explicitly designated to form the edges of the resulting beta-sheets. With all beta-strands preferring to occupy interior (as opposed to edge) locations, intermolecular oligomerization was favored, and the proteins assembled into amyloid-like fibrils. To assess whether explicit design of edge-favoring strands might tip the balance in favor of monomeric beta-sheet proteins, we have now redesigned the first and/or last beta-strands of several sequences from the original library. In the redesigned beta-strands, the binary pattern is changed from [see text] (K denotes lysine). The presence of a lysine on the nonpolar face of a beta-strand should disfavor fibrillar structures because such structures would bury an uncompensated charge. The nonpolar right arrow lysine mutations, therefore, would be expected to favor monomeric structures in which the [see text] sequences form edge strands with the charged lysine side chain accessible to solvent. To test this hypothesis, we constructed several second generation sequences in which the central nonpolar residue of either the N-terminal beta-strand or the C-terminal beta-strand (or both) is changed to lysine. Characterization of the redesigned proteins shows that they form monomeric beta-sheet proteins.
淀粉样纤维与多种神经退行性疾病相关，包括阿尔茨海默病和朊病毒病。淀粉样纤维的结构由与纤维轴垂直排列的β链组成（“交叉β”结构）。我们之前报道了一个从头设计的β折叠蛋白组合文库的设计与表征，这些蛋白能自组装成类似淀粉样的纤维结构。该文库采用“二进制编码”策略设计，其中极性和非极性残基的位置被明确指定，但这些残基的具体身份未作规定且通过组合方式变化。最初的文库设计用于编码包含由反向转角分隔的两亲性β链的蛋白质。每条β链设计为七个残基长，极性（空心圆）和非极性（阴影圆）氨基酸以交替周期性排列（[见图]）。最初的设计为所有β链指定了相同的极性/非极性模式；没有明确指定任何一条链形成所得β折叠的边缘。由于所有β链都倾向于占据内部（而非边缘）位置，分子间寡聚化受到青睐，蛋白质组装成淀粉样纤维。为了评估明确设计倾向于边缘的链是否可能使平衡偏向单体β折叠蛋白，我们现在重新设计了原始文库中几个序列的第一条和/或最后一条β链。在重新设计的β链中，二进制模式从[见图]改变（K表示赖氨酸）。β链非极性面上存在赖氨酸应不利于纤维结构，因为这样的结构会掩埋一个未补偿的电荷。因此，非极性右箭头赖氨酸突变预计会有利于单体结构，其中[见图]序列形成边缘链，带电荷的赖氨酸侧链可接触溶剂。为了验证这一假设，我们构建了几个第二代序列，其中N端β链或C端β链（或两者）的中央非极性残基被改变为赖氨酸。对重新设计的蛋白质的表征表明它们形成单体β折叠蛋白。
