Comparison of NH exchange and circular dichroism as techniques for measuring the parameters of the helix-coil transition in peptides
Comparison of NH exchange and circular dichroism as techniques for measuring the parameters of the helix-coil transition in peptides
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DOI：
10.1021/bi9706677
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发表时间：
1997-07-15
期刊：
影响因子：
2.9
通讯作者：
Baldwin, RL
中科院分区：
文献类型：
--
作者：
Rohl, CA;Baldwin, RL
圆二色性和NH交换直接作为测量肽中螺旋含量和螺旋-卷曲转变的参数的技术进行比较。为了覆盖宽范围的螺旋含量，在0.6至26.9 ℃的温度范围内在1 M氯化钠（H2O）-H-2中检查链长从12至22个残基变化的基于丙氨酸的肽。螺旋-线团转换理论独立地拟合圆二色谱和交换数据，但交换测量的螺旋含量大于圆二色谱测量的螺旋含量。通过去除螺旋中固有的化学交换速率与短的非结构化模型肽测量的交换速率相同的假设，这两种技术达成一致。这种修改允许的圆二色性和NH交换数据被描述由相同的一组螺旋参数，并表明，在螺旋结构的存在下的固有的交换速率降低约17%，相对于在非结构化模型中测得的速率。为了测试这种效应起源于静电的可能性，测定了交换反应对离子强度的敏感性。发现离子强度对交换率有很大的依赖性，但这种依赖性的形式是复杂的。在天然蛋白质的交换速率的研究中，假设蛋白质的交换能力形式以与具有相同氨基酸序列的封闭二肽相同的速率常数进行交换。我们的结果表明，这种假设在某些情况下会严重错误，因为蛋白质中的电荷效应。
Circular dichroism and NH exchange are compared directly as techniques for measuring helix content in peptides and the parameters of the helix-coil transition, To cover a broad range of helix contents, alanine-based peptides with chain lengths varying from 12 to 22 residues are examined over the temperature range from 0.6 to 26.9 degrees C in 1 M sodium chloride, (H2O)-H-2. Helix-coil transition theory independently fits both circular dichroism and exchange data, but the helix contents measured by exchange are larger than those measured by circular dichroism, The two techniques are brought into agreement by removing the assumption that the intrinsic chemical exchange rate in the helix is the same as the exchange rate measured for short unstructured model peptides. This modification allows the circular dichroism and NH exchange data to be described by the same set of helix parameters and indicates that the intrinsic exchange rate in the presence of helical structure is reduced approximately 17% relative to the rates measured in unstructured models. To test the possibility that this effect is electrostatic in origin, the sensitivity of the exchange reaction to ionic strength is determined. A substantial dependence of exchange rate on ionic strength is found, but the form of the dependence is complex. In studies of the exchange rates of native proteins, the exchange-competent form of the protein is assumed to exchange with the same rate constant as a blocked dipeptide with the identical amino acid sequences. Our result suggests that this assumption will be seriously in error in some case because of charge effects in the protein.