Department of Chemistry, University of Illinois at Urbana-Champaign, Urbana, Illinois, USA.
Biophys J. 2011 Jul 20;101(2):421-30. doi: 10.1016/j.bpj.2011.05.071.
We measure the stability and folding relaxation rate of phosphoglycerate kinase (PGK) Förster resonance energy transfer (FRET) constructs localized in the nucleus or in the endoplasmic reticulum (ER) of eukaryotic cells. PGK has a more compact native state in the cellular compartments than in aqueous solution. Its native FRET signature is similar to that previously observed in a carbohydrate-crowding matrix, consistent with crowding being responsible for the compact native state of PGK in the cell. PGK folds through multiple states in vitro, but its folding kinetics is more two-state-like in the ER, so the folding mechanism can be modified by intracellular compartments. The nucleus increases PGK stability and folding rate over the cytoplasm and ER, even though the density of crowders in the nucleus is no greater than in the ER or cytoplasm. Nuclear folding kinetics (and to a lesser extent, thermodynamics) vary less from cell to cell than in the cytoplasm or ER, indicating a more homogeneous crowding and chemical environment in the nucleus.
我们测量了定位于真核细胞核或内质网（ER）中的磷酸甘油酸激酶（PGK）荧光共振能量转移（FRET）构建体的稳定性和折叠松弛速率。PGK 在细胞区室中的天然状态比在水溶液中更紧凑。其天然 FRET 特征与先前在碳水化合物拥挤基质中观察到的特征相似，这表明拥挤是 PGK 在细胞中形成紧凑天然状态的原因。PGK 在体外经历多个折叠状态，但在 ER 中其折叠动力学更类似于两态，因此细胞区室可以修饰折叠机制。与细胞质和 ER 相比，细胞核增加了 PGK 的稳定性和折叠速率，尽管核中拥挤物的密度不比 ER 或细胞质高。细胞核的折叠动力学（以及在较小程度上的热力学）比细胞质或 ER 中的变化更小，表明核中存在更均匀的拥挤和化学环境。
