Biophysics Program, Institute for Physical Science and Technology and Department of Chemistry and Biochemistry, University of Maryland, College Park, MD 20742, USA.
Proc Natl Acad Sci U S A. 2012 Oct 30;109(44):17832-8. doi: 10.1073/pnas.1201808109. Epub 2012 Jul 9.
We use molecular simulations using a coarse-grained model to map the folding landscape of Green Fluorescent Protein (GFP), which is extensively used as a marker in cell biology and biotechnology. Thermal and Guanidinium chloride (GdmCl) induced unfolding of a variant of GFP, without the chromophore, occurs in an apparent two-state manner. The calculated midpoint of the equilibrium folding in GdmCl, taken into account using the Molecular Transfer Model (MTM), is in excellent agreement with the experiments. The melting temperatures decrease linearly as the concentrations of GdmCl and urea are increased. The structural features of rarely populated equilibrium intermediates, visible only in free energy profiles projected along a few order parameters, are remarkably similar to those identified in a number of ensemble experiments in GFP with the chromophore. The excellent agreement between simulations and experiments show that the equilibrium intermediates are stabilized by the chromophore. Folding kinetics, upon temperature quench, show that GFP first collapses and populates an ensemble of compact structures. Despite the seeming simplicity of the equilibrium folding, flux to the native state flows through multiple channels and can be described by the kinetic partitioning mechanism. Detailed analysis of the folding trajectories show that both equilibrium and several kinetic intermediates, including misfolded structures, are sampled during folding. Interestingly, the intermediates characterized in the simulations coincide with those identified in single molecule pulling experiments. Our predictions, amenable to experimental tests, show that MTM is a practical way to simulate the effect of denaturants on the folding of large proteins.
我们使用粗粒度模型的分子模拟来绘制绿色荧光蛋白（GFP）的折叠景观，GFP 在细胞生物学和生物技术中被广泛用作标记物。没有生色团的 GFP 变体的热和盐酸胍（GdmCl）诱导解折叠以明显的两态方式发生。考虑到分子传递模型（MTM），计算得出的 GdmCl 中平衡折叠的中点与实验非常吻合。随着 GdmCl 和尿素浓度的增加，熔点呈线性下降。仅在沿着几个序参数投影的自由能图中可见的稀有平衡中间物的结构特征与具有生色团的 GFP 中的许多集合实验中鉴定的特征非常相似。模拟与实验之间的良好一致性表明平衡中间物被生色团稳定。温度淬火后的折叠动力学表明 GFP 首先坍塌并占据一系列紧凑结构的集合。尽管平衡折叠看似简单，但通量流向天然状态需要通过多个通道，并且可以通过动力学分配机制来描述。对折叠轨迹的详细分析表明，在折叠过程中同时采样了平衡态和几个动力学中间态，包括错误折叠的结构。有趣的是，模拟中表征的中间态与单分子拉伸实验中鉴定的中间态一致。我们的预测，可通过实验测试，表明 MTM 是模拟变性剂对大蛋白折叠影响的一种实用方法。
