White George W N, Gianni Stefano, Grossmann J Gunter, Jemth Per, Fersht Alan R, Daggett Valerie
Biomolecular Structure and Design Program, Department of Medicinal Chemistry, University of Washington, Box 357610, Seattle, WA 98195-7610, USA.
J Mol Biol. 2005 Jul 22;350(4):757-75. doi: 10.1016/j.jmb.2005.05.005.
There is a change from three-state to two-state kinetics of folding across the homeodomain superfamily of proteins as the mechanism slides from framework to nucleation-condensation. The tendency for framework folding in this family correlates with inherent helical propensity. The cellular myeloblastis protein (c-Myb) falls in the mechanistic transition region. An earlier, preliminary report of protein engineering experiments and molecular dynamics simulations (MD) showed that the folding mechanism for this protein has aspects of both the nucleation-condensation and framework models. In the more in-depth analysis of the MD trajectories presented here, we find that folding may be attributed to both of these mechanisms in different regions of the protein. The folding of the loop, middle helix, and turn is best described by nucleation-condensation, whereas folding of the N and C-terminal helices may be described by the framework model. Experimentally, c-Myb folds by apparent two-state kinetics, but the MD simulations predict that the kinetics hide a high-energy intermediate. We stabilized this hypothetical folding intermediate by deleting a residue (P174) in the loop between its second and third helices, and the mutant intermediate is long-lived in the simulations. Equilibrium and kinetic experiments demonstrate that folding of the DeltaP174 mutant is indeed three-state. The presence and shape of the intermediate observed in the simulations were confirmed by small angle X-ray scattering experiments.
随着蛋白质折叠机制从框架模型转变为成核凝聚模型，整个同源结构域超家族的蛋白质折叠动力学发生了从三态到二态的变化。该家族中框架折叠的倾向与固有的螺旋倾向相关。细胞成髓细胞瘤蛋白（c-Myb）处于机制转变区域。一份早期的蛋白质工程实验和分子动力学模拟（MD）的初步报告表明，该蛋白质的折叠机制兼具成核凝聚模型和框架模型的特点。在此处对MD轨迹进行的更深入分析中，我们发现蛋白质的不同区域的折叠可能归因于这两种机制。环、中间螺旋和转角的折叠最好用成核凝聚模型来描述，而N端和C端螺旋的折叠可能用框架模型来描述。实验上，c-Myb以明显的二态动力学进行折叠，但MD模拟预测该动力学隐藏了一个高能中间体。我们通过删除其二螺旋和三螺旋之间环中的一个残基（P174）来稳定这个假设的折叠中间体，并且该突变中间体在模拟中寿命很长。平衡和动力学实验表明，ΔP174突变体的折叠确实是三态的。模拟中观察到的中间体的存在和形状通过小角X射线散射实验得到了证实。
