Institute of Biophysics, Goethe-University Frankfurt, Germany.
Eur Biophys J. 2011 May;40(5):673-85. doi: 10.1007/s00249-011-0673-8. Epub 2011 Jan 28.
The thermal stability and folding dynamics of polyglutamic acid were studied by equilibrium circular dichroism (CD), Fourier-transform infrared (FTIR), and time-resolved temperature-jump infrared (IR) spectroscopy. Polyglutamic acid (PGA) forms α-helical peptides in aqueous solution and is an ideal model system to study the helix-coil transition. Melting curves were monitored with CD and FTIR as a function of pD. At low pD, PGA aggregates at temperatures above 323 K, whereas at pD >5, unfolding and refolding are reversible. At pD 5.4, a helix-coil transition occurs with a transition temperature T(m) of 307 K. At slightly higher pD of 6.2, the peptide conformation is already in a coil structure and only small conformational changes occur upon heating. We determined the equilibrium constant for the reversible helix-coil transition at pD 5.4. The dynamics of this transition was measured at single IR wavelengths after a nanosecond laser-excited temperature jump of ∆T ~ 10 K. Relaxation constants decreased with increasing peptide temperature. Folding and unfolding rates as well as activation energies were extracted based on a two-state model. Our study shows how equilibrium and time-resolved infrared spectroscopic data can be combined to characterize a structural transition and to analyze folding mechanisms.
聚谷氨酸的热稳定性和折叠动力学通过平衡圆二色性（CD）、傅里叶变换红外（FTIR）和时间分辨温度跳跃红外（IR）光谱进行研究。聚谷氨酸（PGA）在水溶液中形成α-螺旋肽，是研究螺旋-卷曲转变的理想模型系统。用 CD 和 FTIR 监测熔融曲线作为 pD 的函数。在低 pD 下，PGA 在高于 323 K 的温度下聚集，而在 pD >5 时，展开和重折叠是可逆的。在 pD 5.4 下，发生螺旋-卷曲转变，转变温度 T(m)为 307 K。在稍微高一点的 pD 6.2 下，肽构象已经处于卷曲结构，只有在加热时才会发生小的构象变化。我们确定了 pD 5.4 下可逆螺旋-卷曲转变的平衡常数。在纳秒激光激发的 ∆T~10 K 的温度跳跃后，在单个 IR 波长下测量了这种转变的动力学。随着肽温度的升高，弛豫常数降低。根据二态模型提取了折叠和展开速率以及活化能。我们的研究表明如何结合平衡和时间分辨红外光谱数据来表征结构转变并分析折叠机制。
