Bédard Sabrina, Mayne Leland C, Peterson Ronald W, Wand A Joshua, Englander S Walter
Johnson Research Foundation, Department of Biochemistry and Biophysics, University of Pennsylvania School of Medicine, Philadelphia, PA 19104-6059, USA.
J Mol Biol. 2008 Feb 29;376(4):1142-54. doi: 10.1016/j.jmb.2007.12.020. Epub 2007 Dec 15.
To search for submolecular foldon units, the spontaneous reversible unfolding and refolding of staphylococcal nuclease under native conditions was studied by a kinetic native-state hydrogen exchange (HX) method. As for other proteins, it appears that staphylococcal nuclease is designed as an assembly of well-integrated foldon units that may define steps in its folding pathway and may regulate some other functional properties. The HX results identify 34 amide hydrogens that exchange with solvent hydrogens under native conditions by way of large transient unfolding reactions. The HX data for each hydrogen measure the equilibrium stability (Delta G(HX)) and the kinetic unfolding and refolding rates (k(op) and k(cl)) of the unfolding reaction that exposes it to exchange. These parameters separate the 34 identified residues into three distinct HX groupings. Two correspond to clearly defined structural units in the native protein, termed the blue and red foldons. The remaining HX grouping contains residues, not well separated by their HX parameters alone, that represent two other distinct structural units in the native protein, termed the green and yellow foldons. Among these four sets, a last unfolding foldon (blue) unfolds with a rate constant of 6 x 10(-6) s(-1) and free energy equal to the protein's global stability (10.0 kcal/mol). It represents part of the beta-barrel, including mutually H-bonding residues in the beta 4 and beta 5 strands, a part of the beta 3 strand that H-bonds to beta 5, and residues at the N-terminus of the alpha2 helix that is capped by beta 5. A second foldon (green), which unfolds and refolds more rapidly and at slightly lower free energy, includes residues that define the rest of the native alpha2 helix and its C-terminal cap. A third foldon (yellow) defines the mutually H-bonded beta1-beta2-beta 3 meander, completing the native beta-barrel, plus an adjacent part of the alpha1 helix. A final foldon (red) includes residues on remaining segments that are distant in sequence but nearly adjacent in the native protein. Although the structure of the partially unfolded forms closely mimics the native organization, four residues indicate the presence of some nonnative misfolding interactions. Because the unfolding parameters of many other residues are not determined, it seems likely that the concerted foldon units are more extensive than is shown by the 34 residues actually observed.
为了寻找亚分子折叠子单元，我们采用动力学天然态氢交换（HX）方法，研究了葡萄球菌核酸酶在天然条件下的自发可逆解折叠和再折叠过程。与其他蛋白质一样，葡萄球菌核酸酶似乎是由整合良好的折叠子单元组装而成，这些单元可能定义了其折叠途径中的步骤，并可能调节其他一些功能特性。HX结果鉴定出34个酰胺氢，它们在天然条件下通过大的瞬时解折叠反应与溶剂氢进行交换。每个氢的HX数据测量了将其暴露于交换的解折叠反应的平衡稳定性（ΔG(HX)）以及动力学解折叠和再折叠速率（k(op)和k(cl)）。这些参数将34个鉴定出的残基分为三个不同的HX分组。其中两个对应于天然蛋白质中明确界定的结构单元，称为蓝色和红色折叠子。其余的HX分组包含仅根据其HX参数无法很好分离的残基，它们代表天然蛋白质中的另外两个不同结构单元，称为绿色和黄色折叠子。在这四组中，最后一个解折叠的折叠子（蓝色）以6×10⁻⁶ s⁻¹的速率常数解折叠，自由能等于蛋白质的整体稳定性（10.0千卡/摩尔）。它代表β桶的一部分，包括β4和β5链中相互形成氢键的残基、与β5形成氢键的β3链的一部分，以及被β5封端的α2螺旋N端的残基。第二个折叠子（绿色）解折叠和再折叠更快，自由能略低，包括定义天然α2螺旋其余部分及其C端封端的残基。第三个折叠子（黄色）定义了相互形成氢键的β1-β2-β3曲折结构，完成了天然β桶，加上α1螺旋的相邻部分。最后一个折叠子（红色）包括序列上相距较远但在天然蛋白质中几乎相邻的其余片段上的残基。尽管部分解折叠形式的结构紧密模仿天然结构，但四个残基表明存在一些非天然的错误折叠相互作用。由于许多其他残基的解折叠参数尚未确定，因此协同折叠子单元可能比实际观察到的34个残基所显示的更为广泛。
