Center for Molecular Protein Science, Biochemistry and Biophysical Chemistry, Lund University, SE-22100 Lund, Sweden.
Proc Natl Acad Sci U S A. 2010 Nov 16;107(46):19826-31. doi: 10.1073/pnas.1005689107. Epub 2010 Nov 1.
Protein stabilization was achieved through in vivo screening based on the thermodynamic linkage between protein folding and fragment complementation. The split GFP system was found suitable to derive protein variants with enhanced stability due to the correlation between effects of mutations on the stability of the intact chain and the effects of the same mutations on the affinity between fragments of the chain. PGB1 mutants with higher affinity between fragments 1 to 40 and 41 to 56 were obtained by in vivo screening of a library of the 1 to 40 fragments against wild-type 41 to 56 fragments. Colonies were ranked based on the intensity of green fluorescence emerging from assembly and folding of the fused GFP fragments. The DNA from the brightest fluorescent colonies was sequenced, and intact mutant PGB1s corresponding to the top three sequences were expressed, purified, and analyzed for stability toward thermal denaturation. The protein sequence derived from the top fluorescent colony was found to yield a 12 °C increase in the thermal denaturation midpoint and a free energy of stabilization of -8.7 kJ/mol at 25 °C. The stability rank order of the three mutant proteins follows the fluorescence rank order in the split GFP system. The variants are stabilized through increased hydrophobic effect, which raises the free energy of the unfolded more than the folded state; as well as substitutions, which lower the free energy of the folded more than the unfolded state; optimized van der Waals interactions; helix stabilization; improved hydrogen bonding network; and reduced electrostatic repulsion in the folded state.
通过基于蛋白质折叠和片段互补之间热力学联系的体内筛选实现蛋白质稳定化。发现分裂 GFP 系统适合衍生出稳定性增强的蛋白质变体，这是由于突变对完整链稳定性的影响与相同突变对链片段之间亲和力的影响之间存在相关性。通过对野生型 41 至 56 片段的 1 至 40 片段文库进行体内筛选，获得了片段 1 至 40 和 41 至 56 之间具有更高亲和力的 PGB1 突变体。根据融合 GFP 片段组装和折叠产生的绿色荧光强度对菌落进行排序。根据最亮荧光菌落的 DNA 进行测序，并表达、纯化和分析对应于前三个序列的完整突变体 PGB1 的稳定性，以抵抗热变性。从最亮荧光菌落衍生出的蛋白质序列产生了 12°C 的热变性中点增加，以及在 25°C 时的-8.7 kJ/mol 的稳定化自由能。三种突变蛋白的稳定性顺序与分裂 GFP 系统中的荧光顺序一致。通过增加疏水性效应来稳定变体，这会提高未折叠状态的自由能超过折叠状态；以及取代，这会降低折叠状态的自由能超过未折叠状态；优化范德华相互作用；稳定螺旋；改善氢键网络；以及在折叠状态下减少静电排斥。
