Tucker Matthew J, Oyola Rolando, Gai Feng
Department of Chemistry, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA.
J Phys Chem B. 2005 Mar 17;109(10):4788-95. doi: 10.1021/jp044347q.
We demonstrate here that a nitrile-derivatized phenylalanine residue, p-cyanophenylalanine (Phe(CN)), and tryptophan (Trp) constitute a novel donor-acceptor pair for fluorescence resonance energy transfer (FRET). The Förster distance of this FRET pair was determined to be approximately 16 A and hence is well suited for determining relatively short separation distances. To validate the applicability of this FRET pair in conformational studies, we studied the conformational heterogeneity of a 14-residue amphipathic peptide, Mastoparan X (MPx peptide), in water and 7 M urea solution as well as at different temperatures. Specifically, seven nitrile-derivatized mutants of the MPx peptide, each containing a Phe(CN) residue that replaces different positions along the peptide sequence (i.e., from position 5 to 11) and serves as a resonance energy donor to the native Trp residue at position 3, were studied spectroscopically. The FRET efficiencies obtained from these peptides allowed us to gain a global picture regarding the conformational distribution of the MPx peptide in different environments. Our results suggest that the MPx molecules exist in water as an ensemble of rather compact conformations, with a radius of gyration of approximately 4.2 A, whereas in 7 M urea the radius of gyration increases to approximately 6.5 A, indicating that the peptide conformations become more extended under this condition. However, we found that temperature had only a negligible effect on the size of the MPx peptide, underlining the difference between the thermally and chemically denatured states of polypeptides. The application of the Gaussian chain or the wormlike chain model allowed us to further obtain the probability distribution function of the separation distance between any two residues along the peptide sequence. We found that the effective bond length of the MPx peptide, obtained by using the Gaussian chain model, is 2.78 A in water and 4.28 A in 7 M urea.
我们在此证明，腈基衍生的苯丙氨酸残基，即对氰基苯丙氨酸（Phe(CN)）和色氨酸（Trp）构成了一种用于荧光共振能量转移（FRET）的新型供体-受体对。该FRET对的福斯特距离经测定约为16 Å，因此非常适合用于确定相对较短的分离距离。为了验证该FRET对在构象研究中的适用性，我们研究了一种14个残基的两亲性肽—— Mastoparan X（MPx肽）在水和7 M尿素溶液中以及在不同温度下的构象异质性。具体而言，对MPx肽的七个腈基衍生突变体进行了光谱研究，每个突变体都含有一个Phe(CN)残基，该残基取代了肽序列中不同位置（即从第5位到第11位）的氨基酸，并作为第3位天然Trp残基的共振能量供体。从这些肽获得的FRET效率使我们能够全面了解MPx肽在不同环境中的构象分布。我们的结果表明，MPx分子在水中以相当紧凑的构象集合形式存在，回转半径约为4.2 Å，而在7 M尿素中回转半径增加到约6.5 Å，这表明在此条件下肽的构象变得更加伸展。然而，我们发现温度对MPx肽的大小影响可忽略不计，这突出了多肽热变性和化学变性状态之间的差异。应用高斯链或蠕虫状链模型使我们能够进一步获得沿肽序列任意两个残基之间分离距离的概率分布函数。我们发现，使用高斯链模型获得的MPx肽的有效键长在水中为2.78 Å，在7 M尿素中为4.28 Å。
