Krishna Mallela M G, Maity Haripada, Rumbley Jon N, Lin Yan, Englander S Walter
Johnson Research Foundation, Department of Biochemistry and Biophysics, University of Pennsylvania School of Medicine, Philadelphia, 19104-6059, USA.
J Mol Biol. 2006 Jun 23;359(5):1410-9. doi: 10.1016/j.jmb.2006.04.035. Epub 2006 May 2.
Previous work used hydrogen exchange (HX) experiments in kinetic and equilibrium modes to study the reversible unfolding and refolding of cytochrome c (Cyt c) under native conditions. Accumulated results now show that Cyt c is composed of five individually cooperative folding units, called foldons, which unfold and refold as concerted units in a stepwise pathway sequence. The first three steps of the folding pathway are linear and sequential. The ordering of the last two steps has been unclear because the fast HX of the amino acid residues in these foldons has made measurement difficult. New HX experiments done under slower exchange conditions show that the final two foldons do not unfold and refold in an obligatory sequence. They unfold separately and neither unfolding obligately contains the other, as indicated by their similar unfolding surface exposure and the specific effects of destabilizing and stabilizing mutations, pH change, and oxidation state. These results taken together support a sequential stabilization mechanism in which folding occurs in the native context with prior native-like structure serving to template the stepwise formation of subsequent native-like foldon units. Where the native structure of Cyt c requires sequential folding, in the first three steps, this is found. Where structural determination is ambiguous, in the final two steps, alternative parallel folding is found.
先前的研究工作利用动力学和平衡模式下的氢交换（HX）实验，在天然条件下研究细胞色素c（Cyt c）的可逆去折叠和重折叠过程。目前积累的结果表明，Cyt c由五个独立协同的折叠单元组成，称为折叠子，它们在逐步的途径序列中作为协同单元去折叠和重折叠。折叠途径的前三个步骤是线性且连续的。最后两个步骤的顺序尚不清楚，因为这些折叠子中氨基酸残基的快速氢交换使得测量变得困难。在较慢交换条件下进行的新氢交换实验表明，最后两个折叠子并非按固定顺序去折叠和重折叠。它们分别去折叠，且一个的去折叠并不必然包含另一个，这从它们相似的去折叠表面暴露以及去稳定化和稳定化突变、pH变化和氧化态的特定影响可以看出。综合这些结果支持了一种顺序稳定机制，即折叠在天然环境中发生，先前类似天然的结构用于模板后续类似天然折叠子单元的逐步形成。在细胞色素c的天然结构需要顺序折叠的地方，在前三个步骤中可以看到这种情况。在结构确定不明确的地方，即最后两个步骤中，发现了交替的平行折叠。
