Microsecond melting of a folding intermediate in a coiled-coil peptide, monitored by T-jump/UV Raman spectroscopy
Microsecond melting of a folding intermediate in a coiled-coil peptide, monitored by T-jump/UV Raman spectroscopy
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DOI：
10.1021/jp061987f
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发表时间：
2006-10-12
影响因子：
3.3
通讯作者：
Spiro, Thomas G.
中科院分区：
文献类型：
--
作者：
Balakrishnan, Gurusamy;Hu, Ying;Spiro, Thomas G.
用197 nm激发的紫外共振拉曼(UVRR)光谱研究了GCN4螺旋卷曲多肽的截断形式，其中酰胺模得到最佳增强。虽然CD的熔化曲线可以用T-m=30℃的两态转变令人满意地描述，但UVRR数据的奇异值分解产生了三个主成分，其温度依赖于折叠和未折叠形式之间的中间形式，形成和熔融温度分别为10℃和40℃。两个α-螺旋酰胺III带，分别在1340和1300 cm(-1)，分别在10℃和40℃选择性融化，并被归类为水化和非水化螺旋区。水合区域被认为以中间形式熔化，而未水合区域是完整的。激光诱导温度跃变后的时间分辨UVRR谱显示出两个弛豫，时间常数分别为0.2u和15u·S，它们可以用来反映水化和非水化螺旋的熔化时间。该非水合螺旋区域可能与已在GCN4的C-末端半部分鉴定的14个残基的“触发”序列有关。螺旋的脱水可能是卷曲线圈折叠的关键步骤。
A truncated version of the GCN4 coiled-coil peptide has been studied by ultraviolet resonance Raman (UVRR) spectroscopy with 197 nm excitation, where amide modes are optimally enhanced. Although the CD melting curve could be satisfactorily described with a two-state transition having T-m = 30 degrees C, singular value decomposition of the UVRR data yielded three principal components, whose temperature dependence implicates an intermediate form between the folded and unfolded forms, with formation and melting temperatures of 10 and 40 degrees C. Two alpha-helical amide III bands, at 1340 and 1300 cm(-1), melted out selectively at 10 and 40 degrees C, respectively, and are assigned to hydrated and unhydrated helical regions. The hydrated regions are proposed to be melted in the intermediate form, while the unhydrated regions are intact. Time-resolved UVRR spectra following laser-induced temperature jumps revealed two relaxations, with time constants of 0.2 and 15 mu s. These are suggested to reflect the melting times of hydrated and unhydrated helices. The unhydrated helical region may be associated with a 14-residue "trigger" sequence that has been identified in the C-terminal half of GCN4. Dehydration of helices may be a key step in the folding of coiled-coils.