Chen L, Wildegger G, Kiefhaber T, Hodgson K O, Doniach S
Department of Chemistry Stanford University, CA 94305, USA.
J Mol Biol. 1998 Feb 13;276(1):225-37. doi: 10.1006/jmbi.1997.1514.
We report time-resolved small angle X-ray scattering (SAXS) studies of the structural characteristics of the collapsed state of lysozyme from henegg white (HEL) obtained on initiating refolding by rapidly changing solvent conditions from 8 M to 1.1 M urea at pH 2.9. At this reduced pH the lifetime, of about one second, of the non-specifically collapsed ensemble is considerably prolonged relative to its value at pH 5.2. The SAXS studies are combined with time resolved measurements of tryptophan fluorescence and of the rate of formation of native molecules using interrupted refolding experiments. We observe large burst phase changes in intrinsic tryptophan fluorescence and in the radius of gyration (Rg) which is reduced from 22 A in the fully unfolded state to approximately 19 to 20 A. Subsequent decrease of the Rg to the value for native lysozyme (15 A) follows the time course of formation of native molecules. Single exponential fits to the singular value decomposition (SVD) components of the SAXS data allow reconstruction of the SAXS profile at early time points of refolding. The results of this analysis suggest a globular shape of the collapsed state. A similar fit to the forward scattering amplitude, I(0), suggests that the collapsed state has a solvent accessible surface area which is considerably increased relative to that of the native protein. These results show directly that the non-specifically collapsed state formed during the burst phase in lysozyme refolding indeed represents a molecular compaction and a change in shape from a fully denatured random coil state (albeit restricted by disulfide bonds) to an ensemble of globular conformations which, however, have not yet formed a solvent-protected hydrophobic core.
我们报告了时间分辨小角X射线散射（SAXS）研究结果，该研究针对在pH 2.9条件下通过将溶剂条件从8 M尿素快速改变为1.1 M尿素来引发重折叠时获得的鸡蛋清溶菌酶（HEL）折叠态的结构特征。在这个较低的pH值下，非特异性折叠聚集体约一秒的寿命相对于其在pH 5.2时的值显著延长。SAXS研究与色氨酸荧光的时间分辨测量以及使用间断重折叠实验对天然分子形成速率的测量相结合。我们观察到内在色氨酸荧光和回转半径（Rg）在爆发阶段有大幅变化，回转半径从完全展开状态下的22 Å减小到约19至20 Å。随后Rg减小到天然溶菌酶的值（15 Å）遵循天然分子形成的时间进程。对SAXS数据的奇异值分解（SVD）分量进行单指数拟合，能够在重折叠的早期时间点重建SAXS轮廓。该分析结果表明折叠态具有球状形状。对前向散射振幅I(0)进行类似拟合表明，折叠态具有相对于天然蛋白质显著增加的溶剂可及表面积。这些结果直接表明，溶菌酶重折叠爆发阶段形成的非特异性折叠态确实代表了一种分子压缩以及形状从完全变性的无规卷曲状态（尽管受二硫键限制）转变为球状构象聚集体，然而，这些构象尚未形成溶剂保护的疏水核心。
