Department of Physics, Chemical and Biomolecular Engineering, and Chemistry and Center for Biophysics and Computational Biology, University of Illinois, Urbana, IL 61801, USA.
Proc Natl Acad Sci U S A. 2012 Oct 30;109(44):17863-7. doi: 10.1073/pnas.1201797109. Epub 2012 Jun 4.
We measure the stability and folding rate of a mutant of the enzyme phosphoglycerate kinase (PGK) inside bone tissue cells as a function of temperature from 38 to 48 °C. To facilitate measurement in individual living cells, we developed a rapid laser temperature stepping method capable of measuring complete thermal melts and kinetic traces in about two min. We find that this method yields improved thermal melts compared to heating a sample chamber or microscope stage. By comparing results for six cells with in vitro data, we show that the protein is stabilized by about 6 kJ/mole in the cytoplasm, but the temperature dependence of folding kinetics is similar to in vitro. The main difference is a slightly steeper temperature dependence of the folding rate in some cells that can be rationalized in terms of temperature-dependent crowding, local viscosity, or hydrophobicity. The observed rate coefficients can be fitted within measurement uncertainty by an effective two-state model, even though PGK folds by a multistate mechanism. We validate the effective two-state model with a three-state free energy landscape of PGK to illustrate that the effective fitting parameters can represent a more complex underlying free energy landscape.
我们测量了作为温度函数的骨组织细胞内磷酸甘油酸激酶 (PGK) 突变体的稳定性和折叠率，温度范围为 38 至 48°C。为了便于在单个活细胞中进行测量，我们开发了一种快速激光温度阶跃方法，能够在大约两分钟内测量完整的热融解和动力学轨迹。我们发现，与加热样品室或显微镜载物台相比，这种方法可以得到更好的热融解结果。通过将六个细胞的结果与体外数据进行比较，我们表明该蛋白在细胞质中稳定约 6 kJ/mol，但折叠动力学的温度依赖性与体外相似。主要区别在于一些细胞中折叠速率的温度依赖性略高，这可以根据温度依赖性拥挤、局部粘度或疏水性来合理化。观察到的速率系数可以在测量不确定度内通过有效两态模型拟合，即使 PGK 通过多态机制折叠。我们使用 PGK 的三态自由能景观验证了有效两态模型，以说明有效拟合参数可以代表更复杂的潜在自由能景观。
