Dantas Gautam, Kuhlman Brian, Callender David, Wong Michelle, Baker David
Department of Biochemistry, University of Washington, Seattle, WA 98195, USA.
J Mol Biol. 2003 Sep 12;332(2):449-60. doi: 10.1016/s0022-2836(03)00888-x.
A previously developed computer program for protein design, RosettaDesign, was used to predict low free energy sequences for nine naturally occurring protein backbones. RosettaDesign had no knowledge of the naturally occurring sequences and on average 65% of the residues in the designed sequences differ from wild-type. Synthetic genes for ten completely redesigned proteins were generated, and the proteins were expressed, purified, and then characterized using circular dichroism, chemical and temperature denaturation and NMR experiments. Although high-resolution structures have not yet been determined, eight of these proteins appear to be folded and their circular dichroism spectra are similar to those of their wild-type counterparts. Six of the proteins have stabilities equal to or up to 7kcal/mol greater than their wild-type counterparts, and four of the proteins have NMR spectra consistent with a well-packed, rigid structure. These encouraging results indicate that the computational protein design methods can, with significant reliability, identify amino acid sequences compatible with a target protein backbone.
一个先前开发的用于蛋白质设计的计算机程序RosettaDesign，被用于预测九条天然存在的蛋白质主链的低自由能序列。RosettaDesign并不知晓天然存在的序列，设计序列中平均65%的残基与野生型不同。生成了十个完全重新设计的蛋白质的合成基因，这些蛋白质被表达、纯化，然后使用圆二色性、化学和温度变性以及核磁共振实验进行表征。尽管尚未确定高分辨率结构，但其中八个蛋白质似乎已折叠，并且它们的圆二色性光谱与野生型对应物的光谱相似。六个蛋白质的稳定性比其野生型对应物相等或高出多达7千卡/摩尔，并且四个蛋白质的核磁共振光谱与紧密堆积、刚性的结构一致。这些令人鼓舞的结果表明，计算蛋白质设计方法能够以显著的可靠性识别与目标蛋白质主链兼容的氨基酸序列。
