Skelton N J, Garcia K C, Goeddel D V, Quan C, Burnier J P
Department of Protein Engineering, Genentech, Inc., South San Francisco, California 94080.
Biochemistry. 1994 Nov 22;33(46):13581-92. doi: 10.1021/bi00250a010.
Guanylin is a 15 amino acid mammalian hormone containing two disulfide bonds. Guanylin shares sequence similarity with the bacterial heat-stable enterotoxin (STa) and is capable of binding to and stimulating the STa guanylyl cyclase receptor. Biologically active peptides have been prepared by two methods: (1) enzymatic treatment of a 99 residue proprotein (denoted proguanylin) expressed in Escherichia coli and (2) solid-phase chemical synthesis. Although both sources yield material that is pure by high-performance liquid chromatography and mass spectrometry, analysis by nuclear magnetic resonance (NMR) indicates that peptides from both sources contain two conformationally distinct species present in a 1:1 ratio. The chemical shift differences between the two species are large, allowing unambiguous sequential NMR assignments to be made for both sets of resonances. Exchange between the two forms was not observed even at 70 degrees C. Structural restraints have been generated from nuclear Overhauser effects and scalar coupling constants and used to calculate structures for both forms using distance geometry and restrained energy minimization. The resulting structures for the first isoform are well defined (root-mean-square deviation from the average structure for backbone atoms of 0.47 A) and adopt a right-handed spiral conformation, similar to that observed for heat stable enterotoxin. The second isoform is less well defined (root-mean-square deviation from the average structure for backbone atoms of 1.07 A) but clearly adopts a very different fold consisting of a left-hand spiral. The differences in structure suggest that the two forms may have very different affinities toward the STa receptor. The observation of such isomerism has important implications for the common practice of introducing multiple disulfide bonds into small peptides to limit conformational flexibility and enhance bioactivity.
鸟苷林是一种含有两个二硫键的由15个氨基酸组成的哺乳动物激素。鸟苷林与细菌热稳定肠毒素（STa）具有序列相似性，能够结合并刺激STa鸟苷酸环化酶受体。生物活性肽通过两种方法制备：（1）对在大肠杆菌中表达的99个残基的前体蛋白（称为前鸟苷林）进行酶处理；（2）固相化学合成。尽管两种来源产生的物质通过高效液相色谱和质谱分析均为纯品，但核磁共振（NMR）分析表明，两种来源的肽均包含两种构象不同的物种，且比例为1:1。这两种物种之间的化学位移差异很大，使得可以对两组共振进行明确的序列NMR归属。即使在70摄氏度下也未观察到两种形式之间的交换。已从核Overhauser效应和标量耦合常数生成结构限制，并用于使用距离几何和受限能量最小化方法计算两种形式的结构。第一种异构体的最终结构定义明确（主链原子的平均结构的均方根偏差为0.47 Å），并采用右手螺旋构象，类似于热稳定肠毒素所观察到的构象。第二种异构体的定义不太明确（主链原子的平均结构的均方根偏差为1.07 Å），但明显采用了由左手螺旋组成的非常不同的折叠方式。结构上的差异表明这两种形式对STa受体可能具有非常不同的亲和力。这种异构现象的观察对于在小肽中引入多个二硫键以限制构象灵活性并增强生物活性的常见做法具有重要意义。
