Rescue of degradation-prone mutants of the FK506-rapamycin binding (FRB) protein with chemical ligands
Rescue of degradation-prone mutants of the FK506-rapamycin binding (FRB) protein with chemical ligands
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DOI：
10.1002/cbic.200700087
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发表时间：
2007-07-09
期刊：
影响因子：
3.2
通讯作者：
Gestwicki, Jason E.
中科院分区：
文献类型：
--
作者：
Stankunas, Kryn;Bayle, J. Henri;Gestwicki, Jason E.
我们最近报道了FK 506-雷帕霉素结合（FRB）结构域中的某些突变破坏其体外和体内稳定性（Stankunas等人，Mol. Cell，2003，12，7615）。为了确定引起不稳定性的精确残基，我们通过测量可逆离液变性期间FRB突变体的固有色氨酸荧光来计算FRB突变体的集合的折叠自由能（Δ G）。我们的研究结果暗示T2098 L点突变是不稳定性的关键决定因素。此外，我们发现，在这个集合中的一些突变体是不稳定的高达6 kcal mol（-1）相对于野生型。为了研究这些突变体在细胞中的行为，我们在非洲绿色猴肾（CO-S）细胞系和小鼠胚胎成纤维细胞（MEF）中表达了与FRB突变体融合的萤火虫荧光素酶。当使用不稳定的FRB突变体时，我们发现蛋白质水平和发光强度都很低。然而，添加FRB的化学配体雷帕霉素恢复了荧光素酶活性。有趣的是，我们发现体外计算的FRB突变体的Δ G与细胞中的相对化学拯救之间存在大致线性关系。由于雷帕霉素能够同时结合FRB和伴侣FK 506结合蛋白（FKBP），我们接下来检查FKBP是否有助于保护FRB突变体。使用体外实验和基于细胞的模型，我们发现FKBP稳定突变体。这些发现与最近的模型一致，该模型表明对内在Delta G的损伤可以通过药理学伴侣来纠正。此外，这些结果提供了用于控制蛋白质稳定性的条件稳定的融合配偶体的集合。
We recently reported that certain mutations in the FK506-rapamycin binding (FRB) domain disrupt its stability in vitro and in vivo (Stankunas et al. Mol. Cell, 2003, 12, 7615). To determine the precise residues that cause instability, we calculated the folding free energy (Delta G) of a collection of FRB mutants by measuring their intrinsic tryptophan fluorescence during reversible chaotropic denaturation. Our results implicate the T2098L point mutation as a key determinant of instability. Further, we found that some of the mutants in this collection were destabilised by up to 6 kcal mol(-1) relative to the wild type. To investigate how these mutants behave in cells, we expressed firefly luciferase fused to FRB mutants in African green monkey kidney (CO-S) cell lines and mouse embryonic fibroblasts (MEFs). When unstable FRB mutants were used, we found that the protein levels and the luminescence intensities were low. However, addition of a chemical ligand for FRB, rapamycin, restored luciferase activity. Interestingly, we found a roughly linear relationship between the Delta G of the FRB mutants calculated in vitro and the relative chemical rescue in cells. Because rapamycin is capable of simultaneously binding both FRB and the chaperone, FK506-binding protein (FKBP), we next examined whether FKBP might contribute to the protection of FRB mutants. Using both in vitro experiments and a cell-based model, we found that FKBP stabilizes the mutants. These findings are consistent with recent models that suggest damage to intrinsic Delta G can be corrected by pharmacological chaperones. Further, these results provide a collection of conditionally stable fusion partners for use in controlling protein stability.