Engineered metal binding sites map the heterogeneous folding landscape of a coiled coil
Engineered metal binding sites map the heterogeneous folding landscape of a coiled coil
复制标题
DOI：
10.1038/nsb723
复制
发表时间：
2001-12-01
期刊：
影响因子：
--
通讯作者：
Sosnick, TR
中科院分区：
文献类型：
--
作者：
Krantz, BA;Sosnick, TR
为了解决蛋白质是否沿着多个途径折叠，i,i+4 双组氨酸金属结合位点被引入生长控制转录因子 GCN4 的亮氨酸拉链区域的二聚体和交联版本中。二价金属离子结合增强了 GCN4 的平衡和折叠活化自由能。折叠率的增强量化了在过渡态下具有螺旋几何结构的结合位点的分子的分数。因此，这种称为 Psi 分析的新方法可以识别以两种状态方式折叠的蛋白质的途径异质性程度，这种能力即使使用单分子方法也通常无法实现。不断调整金属离子浓度可以改变双组氨酸区域的稳定性，而不会对蛋白质造成额外的结构扰动。对于二聚体和交联版本，每个金属离子浓度下动力学势垒高度的伴随变化绘制了折叠景观，并确定了连接在路径选择中的重要性。此外，该方法可以推广到其他生物物理研究，其中连续调整特定区域的稳定性而没有无关的结构扰动的能力是有利的。
To address whether proteins fold along multiple pathways, i,i+4 bi-histidine metal binding sites are introduced into dimeric and crosslinked versions of the leucine zipper region of the growth control transcription factor, GCN4. Divalent metal ion binding enhances both the equilibrium and folding activation free energies for GCN4. The enhancement of folding rates quantifies the fraction of molecules that have the binding site in a helical geometry in the transition state. Hence, this new method, termed Psi -analysis, identifies the degree of pathway heterogeneity for a protein that folds in a two-state manner, a capability that is generally unavailable even with single molecule methods. Adjusting metal ion concentration continuously varies the stability of the bi-histidine region without additional structural perturbation to the protein. For dimeric and crosslinked versions, the accompanying changes in kinetic barrier heights at each metal ion concentration maps the folding landscape as well as establishes the importance of connectivity in pathway selection. Furthermore, this method can be generalized to other biophysical studies, where the ability to continuously tune the stability of a particular region with no extraneous structural perturbation is advantageous.