Finke John M, Jennings Patricia A, Lee Jennifer C, Onuchic José N, Winkler Jay R
Department of Chemistry, Oakland University, Rochester, MI 48309-4477, USA.
Biopolymers. 2007 Jun 15;86(3):193-211. doi: 10.1002/bip.20719.
The equilibrium structural ensemble of a 20-residue polyglutamic acid peptide (E(20)) was studied with FRET, circular dichroism, and molecular dynamics (MD) simulations. A FRET donor, o-aminobenzamide, and acceptor, 3-nitrotyrosine, were introduced at the N- and C-termini, respectively. Circular dichroism, steady state FRET, and time-resolved FRET measurements were employed to characterize the fraction helix and end-to-end distance under different pH conditions: pH 4 (60% alpha-helix), pH 6 (0% alpha-helix), and pH 9 (0% alpha-helix). At pH 4, the end-to-end distance was measured at 24 A and determined to be considerably less than the 31 A predicted for an alpha-helix of the same length. At pH 6 and 9, the end-to-end distance was measured at > 31 and 39 A respectively, both which are determined to be considerably greater than the 27 A predicted for a freely jointed random coil of the same length. To better understand the physical forces underlying the unusual helix-coil transition in this peptide, three theoretical MD models of E(20) were constructed: (1) a pure alpha-helix, (2) an alpha-helix with equivalent attractive intramolecular contacts, and (3) a weak alpha-helix with termini-weighted intramolecular contacts ("sticky ends"). Using MD simulations, the bent helix structure calculated from Model 3 was found to be the closest in agreement with the experimental data.
利用荧光共振能量转移（FRET）、圆二色性和分子动力学（MD）模拟研究了一种20个残基的聚谷氨酸肽（E(20)）的平衡结构集合。分别在N端和C端引入了FRET供体邻氨基苯甲酰胺和受体3-硝基酪氨酸。采用圆二色性、稳态FRET和时间分辨FRET测量来表征不同pH条件下的螺旋分数和端到端距离：pH 4（60%α-螺旋）、pH 6（0%α-螺旋）和pH 9（0%α-螺旋）。在pH 4时，端到端距离测量为24 Å，确定明显小于相同长度α-螺旋预测的31 Å。在pH 6和9时，端到端距离分别测量为>31 Å和39 Å，两者都确定明显大于相同长度自由连接随机卷曲预测的27 Å。为了更好地理解该肽中异常螺旋-卷曲转变背后的物理力，构建了E(20)的三个理论MD模型：（1）纯α-螺旋，（2）具有等效吸引分子内接触的α-螺旋，以及（3）具有末端加权分子内接触（“粘性末端”）的弱α-螺旋。使用MD模拟，发现从模型3计算出的弯曲螺旋结构与实验数据最吻合。
