Department of Biochemistry, Robert Wood Johnson Medical School, UMDNJ, and Center for Advanced Biotechnology and Medicine, Piscataway, New Jersey 08854, USA.
Biochemistry. 2010 Mar 23;49(11):2307-16. doi: 10.1021/bi902077d.
We sought to computationally design model collagen peptides that specifically associate as heterotrimers. Computational design has been successfully applied to the creation of new protein folds and functions. Despite the high abundance of collagen and its key role in numerous biological processes, fibrous proteins have received little attention as computational design targets. Collagens are composed of three polypeptide chains that wind into triple helices. We developed a discrete computational model to design heterotrimer-forming collagen-like peptides. Stability and specificity of oligomerization were concurrently targeted using a combined positive and negative design approach. The sequences of three 30-residue peptides, A, B, and C, were optimized to favor charge-pair interactions in an ABC heterotrimer, while disfavoring the 26 competing oligomers (i.e., AAA, ABB, BCA). Peptides were synthesized and characterized for thermal stability and triple-helical structure by circular dichroism and NMR. A unique A:B:C-type species was not achieved. Negative design was partially successful, with only A + B and B + C competing mixtures formed. Analysis of computed versus experimental stabilities helps to clarify the role of electrostatics and secondary-structure propensities determining collagen stability and to provide important insight into how subsequent designs can be improved.
我们试图通过计算设计专门作为异三聚体结合的模型胶原蛋白肽。计算设计已成功应用于新蛋白质折叠和功能的创造。尽管胶原蛋白含量丰富，在许多生物过程中起着关键作用，但纤维状蛋白质作为计算设计目标受到的关注较少。胶原蛋白由三条多肽链组成，这些多肽链缠绕成三螺旋。我们开发了一种离散的计算模型来设计形成异三聚体的胶原蛋白样肽。使用正设计和负设计相结合的方法，同时针对寡聚化的稳定性和特异性。优化了三个 30 个残基的肽 A、B 和 C 的序列，以有利于 ABC 异三聚体中的电荷对相互作用，同时不利于 26 种竞争寡聚物（即 AAA、ABB、BCA）。通过圆二色性和 NMR 对合成的肽进行了热稳定性和三螺旋结构的表征。未获得独特的 A:B:C 型物质。负设计部分成功，仅形成 A+B 和 B+C 的竞争混合物。计算稳定性与实验稳定性的分析有助于阐明静电和二级结构倾向决定胶原蛋白稳定性的作用，并为如何改进后续设计提供重要的见解。
