Residue specific resolution of protein folding dynamics using isotope-edited infrared temperature jump spectroscopy
Residue specific resolution of protein folding dynamics using isotope-edited infrared temperature jump spectroscopy
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DOI：
10.1021/bi602372y
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发表时间：
2007-03-20
期刊：
影响因子：
2.9
通讯作者：
Dyer, R. Brian
中科院分区：
文献类型：
--
作者：
Brewer, Scott H.;Song, Benben;Dyer, R. Brian
蛋白质折叠实验研究的一个主要困难是缺乏非扰动的、残基特异性的折叠探针。在这里，我们证明了使用（CO）-C-13-O-18同位素编辑红外光谱在单个残基水平上解析蛋白质折叠动力学的能力。一个单一的（CO）-C-13-O-18同位素标记掺入到36个残基的三螺旋束绒毛蛋白头片段亚结构域（HP 36）的骨架中。将标记物置于蛋白质的第二个α-螺旋的溶剂保护区中。（CO）-C-13-O-18同位素标记使折叠状态下的羰基伸缩频率移动到1572.1 cm（-1），与未标记蛋白质骨架的（CO）-C-12-O-16带完全分离。利用（CO）-C-13-O-18标记的独特IR特征，使用温度依赖性FTIR光谱来探测平衡热去折叠转变。使用温度跳跃（T-jump）IR光谱监测折叠/展开动力学。平衡展开的研究表明，构象的变化，提示在螺旋2的螺旋结构的损失之前，全球展开的蛋白质。探测标记位点和（CO）-C-12-O-16带的T-跳跃弛豫动力学被发现是具有相似弛豫速率的双相的。慢弛豫相（类似于2 × 10（5）s（-1））对应于全局折叠转变。标记的位置，螺旋2中的一个埋藏位置，提供了快速弛豫相（类似于10（7）s（-1））起源的重要探针。该相对于标记的位置具有显著的振幅，即使其在折叠结构中被很好地保护免受溶剂的影响。快速相可能代表快速预平衡，其涉及标记周围的溶剂渗透和在全局解折叠转变之前螺旋2的可能部分解折叠。这项工作是第一次实验研究的超快折叠动力学与残基特定的分辨率。
A major difficulty in experimental studies of protein folding is the lack of nonperturbing, residue specific probes of folding. Here, we demonstrate the ability to resolve protein folding dynamics at the level of a single residue using (CO)-C-13-O-18 isotope-edited infrared spectroscopy. A single (CO)-C-13-O-18 isotopic label was incorporated into the backbone of the 36 residue, three-helix bundle villin headpiece subdomain (HP36). The label was placed in a solvent protected region of the second alpha-helix of the protein. The (CO)-C-13-O-18 isotopic label shifted the carbonyl stretching frequency to 1572.1 cm(-1) in the folded state, well removed from the (CO)-C-12-O-16 band of the unlabeled protein backbone. The unique IR signature of the (CO)-C-13-O-18 label was exploited to probe the equilibrium thermal unfolding transition using temperature-dependent FTIR spectroscopy. The folding/unfolding dynamics were monitored using temperature-jump (T-jump) IR spectroscopy. The equilibrium unfolding studies showed conformational changes suggestive of a loss of helical structure in helix 2 prior to the global unfolding of the protein. T-jump relaxation kinetics probing both the labeled site and the (CO)-C-12-O-16 band were found to be biphasic with similar relaxation rates. The slow relaxation phase (similar to 2 x 10(5) s(-1)) corresponds to the global folding transition. The location of the label, a buried position in helix 2, provides an important probe of the origin of the fast relaxation phase (similar to 10(7) s(-1)). This phase has significant amplitude for the labeled position even though it is well protected from solvent in the folded structure. The fast phase likely represents a rapid pre-equilibrium that involves solvent penetration around the label and possible partial unfolding of helix 2 prior to the global unfolding transition. This work represents the first experimental study of ultrafast folding dynamics with residue specific resolution.