Department of Chemistry, CB 3290, University of North Carolina, Chapel Hill, North Carolina 27599, United States.
Biochemistry. 2011 Apr 5;50(13):2575-84. doi: 10.1021/bi101116a. Epub 2011 Mar 10.
A β-sheet miniprotein based on the FBP11 WW1 domain sequence has been redesigned for the molecular recognition of ssDNA. A previous report showed that a β-hairpin peptide dimer, (WKWK)(2), binds ssDNA with low micromolar affinity but with little selectivity over duplex DNA. This report extends those studies to a three-stranded β-sheet miniprotein designed to mimic the OB-fold. The new peptide binds ssDNA with low micromolar affinity and shows about 10-fold selectivity for ssDNA over duplex DNA. The redesigned peptide no longer binds its native ligand, the polyproline helix, confirming that the peptide has been redesigned for the function of binding ssDNA. Structural studies provide evidence that this peptide consists of a well-structured β-hairpin made of strands 2 and 3 with a less structured first strand that provides affinity for ssDNA but does not improve the stability of the full peptide. These studies provide insight into protein-DNA interactions as well as a novel example of protein redesign.
基于 FBP11 WW1 结构域序列的 β-折叠片小蛋白已被重新设计用于 ssDNA 的分子识别。先前的报告表明，β发夹肽二聚体（WKWK）（2）以低微摩尔亲和力结合 ssDNA，但对双链 DNA 几乎没有选择性。本报告将这些研究扩展到了一种设计成模拟 OB 折叠的三股β折叠片小蛋白。新肽以低微摩尔亲和力结合 ssDNA，并显示出约 10 倍的 ssDNA 对双链 DNA 的选择性。重新设计的肽不再结合其天然配体，即多聚脯氨酸螺旋，这证实了该肽已被重新设计用于结合 ssDNA 的功能。结构研究提供了证据，证明该肽由由链 2 和 3 组成的结构良好的 β 发夹组成，具有结构较差的第一链，该链提供了与 ssDNA 的亲和力，但不能提高全长肽的稳定性。这些研究为蛋白质-DNA 相互作用提供了深入的了解，以及蛋白质重新设计的一个新范例。
