Department of Chemistry, York University, 4700 Keele Street, Toronto, Ontario, Canada M3J 1P3.
Biochemistry. 2010 Oct 5;49(39):8478-87. doi: 10.1021/bi100952k. Epub 2010 Sep 7.
We have used a combined approach of NMR spectroscopy and isothermal titration calorimetry (ITC) to determine the ligand-binding mechanism employed by a cocaine-binding aptamer. We found that the length of the stem containing the 3' and 5' termini determines the nature of the binding mechanism. When this stem is six base pairs long, the secondary structure of the aptamer is fully folded in the free form and only putative tertiary interactions form with ligand binding. If this stem is shortened by three base pairs, the free form of the aptamer contains little secondary structure, and ligand binding triggers secondary structure formation and folding. This binding mechanism is supported by both NMR spectral changes and the ITC measured heat capacity of binding (ΔC(p)°). For the aptamer with the long stem the ΔC(p)° value is -557 ± 29 cal mol(-1) K(-1) and for the aptamer with the short stem the ΔC(p)° value is -922 ± 51 cal mol(-1) K(-1). Chemical shift perturbation data and the observation of intermolecular NOEs indicate that the three-way junction is the site of ligand binding.
我们采用 NMR 光谱和等温热滴定（ITC）相结合的方法，确定了可卡因结合适体所采用的配体结合机制。我们发现，包含 3' 和 5' 末端的茎的长度决定了结合机制的性质。当这个茎长为六个碱基对时，适体的二级结构在游离形式下完全折叠，只有假定的三级相互作用与配体结合。如果这个茎缩短三个碱基对，适体的游离形式几乎没有二级结构，配体结合触发二级结构的形成和折叠。这一结合机制得到了 NMR 光谱变化和 ITC 测量的结合热容（ΔC(p)°）的支持。对于长茎适体，ΔC(p)°值为-557 ± 29 cal mol(-1) K(-1)，而对于短茎适体，ΔC(p)°值为-922 ± 51 cal mol(-1) K(-1)。化学位移扰动数据和观察到的分子间 NOE 表明，三链结是配体结合的位点。
