Department of Chemistry, University of Wyoming , Laramie, Wyoming 82071, United States.
J Am Chem Soc. 2014 Apr 23;136(16):6037-48. doi: 10.1021/ja500918k. Epub 2014 Apr 15.
The mechanism of protein folding remains poorly understood, in part due to limited experimental information available about partially folded states. Isotopically edited infrared (IR) spectroscopy has emerged as a promising method for studying protein structural changes with site-specific resolution, but its full potential to systematically probe folding at multiple protein sites has not yet been realized. We have used (13)C isotopically edited IR spectroscopy to investigate the site-specific thermal unfolding at seven different locations in the de novo designed helix-turn-helix protein αtα. As one of the few stable helix-turn-helix motifs, αtα is an excellent model for studying the roles of secondary and tertiary interactions in folding. Circular dichroism (CD) experiments on the full αtα motif and its two peptide fragments show that interhelical tertiary contacts are critical for stabilization of the secondary structure. The site-specific thermal unfolding probed by (13)C isotopically edited IR is likewise consistent with primarily tertiary stabilization of the local structure. The least thermally stable part of the αtα motif is near the turn where the interhelical contacts are rather loose, while the motif's center with best established core packing has the highest stability. Similar correlation between the local thermal stability and tertiary contacts was found previously for a naturally occurring helix-turn-helix motif. These results underline the importance of native-like tertiary stabilizing interactions in folding, in agreement with recent state-of-the art folding simulations as well as simplified, native-centric models.
蛋白质折叠的机制仍未被充分理解，部分原因是关于部分折叠状态的实验信息有限。同位素编辑的红外（IR）光谱学已成为一种有前途的方法，可以对蛋白质结构变化进行具有特定部位分辨率的研究，但尚未充分发挥其对多个蛋白质部位折叠进行系统探测的潜力。我们使用（13）C 同位素编辑的红外光谱学研究了从头设计的螺旋-转角-螺旋蛋白αtα 中七个不同位置的特定部位热解折叠。作为少数几个稳定的螺旋-转角-螺旋基序之一，αtα 是研究二级和三级相互作用在折叠中作用的理想模型。全αtα 基序及其两个肽片段的圆二色性（CD）实验表明，螺旋间的三级接触对于稳定二级结构至关重要。（13）C 同位素编辑的 IR 探测到的特定部位热解折叠同样与局部结构的主要三级稳定一致。αtα 基序中最不稳定的部分靠近转角，那里的螺旋间接触比较松散，而具有最佳核心包装的基序中心则具有最高的稳定性。先前在天然存在的螺旋-转角-螺旋基序中也发现了局部热稳定性与三级接触之间的类似相关性。这些结果强调了天然样三级稳定相互作用在折叠中的重要性，与最近的最先进的折叠模拟以及简化的、以天然为中心的模型一致。
