Buscaglia Marco, Lapidus Lisa J, Eaton William A, Hofrichter James
Laboratory of Chemical Physics, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892-0520, USA.
Biophys J. 2006 Jul 1;91(1):276-88. doi: 10.1529/biophysj.105.071167. Epub 2006 Apr 14.
Quenching of the triplet state of tryptophan by close contact with cysteine has been used to measure the reaction-limited and diffusion-limited rates of loop formation in disordered polypeptides having the sequence cys-(ala-gly-gln)j-trp (j=1-9). The decrease in the length-dependence of the reaction-limited rate for short chains in aqueous buffer, previously attributed to chain stiffness, is not observed at high concentrations of chemical denaturant (6 M GdmCl and 8 M urea), showing that denaturants increase chain flexibility. For long chains, both reaction-limited and diffusion-limited rates are significantly smaller in denaturant and exhibit a steeper length dependence. The results can be explained using end-to-end distributions from a wormlike chain model in which excluded volume interactions are incorporated by associating a 0.4-0.5 nm diameter hard sphere with the end of each virtual peptide bond. Fitting the data with this model shows that the denaturants reduce the persistence length from approximately 0.6 nm to approximately 0.4 nm, only slightly greater than the length of a peptide bond. The same model also describes the reported length dependence for the radii of gyration of chemically denatured proteins containing 50-400 residues. The end-to-end diffusion coefficients obtained from the diffusion-limited rates are smaller than the sum of the monomer diffusion coefficients and exhibit significant temperature dependence, suggesting that diffusion is slowed by internal friction arising from barriers to backbone conformational changes.
通过与半胱氨酸紧密接触淬灭色氨酸的三重态，已被用于测量具有序列cys-(ala-gly-gln)j-trp（j = 1 - 9）的无序多肽中环形成的反应限制速率和扩散限制速率。在水缓冲液中，短链的反应限制速率的长度依赖性降低，此前归因于链的刚性，但在高浓度化学变性剂（6 M盐酸胍和8 M尿素）存在下未观察到这种情况，这表明变性剂增加了链的柔韧性。对于长链，变性剂中的反应限制速率和扩散限制速率都显著较小，并且表现出更陡峭的长度依赖性。使用蠕虫状链模型的端到端分布可以解释这些结果，其中通过将直径为0.4 - 0.5 nm的硬球与每个虚拟肽键的末端相关联来纳入排除体积相互作用。用该模型拟合数据表明，变性剂将持久长度从约0.6 nm降低到约0.4 nm，仅略大于一个肽键的长度。相同的模型还描述了报道的含有50 - 400个残基的化学变性蛋白质的回转半径的长度依赖性。从扩散限制速率获得的端到端扩散系数小于单体扩散系数之和，并且表现出显著的温度依赖性，这表明扩散因主链构象变化的障碍引起的内摩擦而减慢。
