NSF Center for Biophotonics Science and Technology, School of Medicine, University of California Davis, Sacramento, California 95817, USA.
Protein Sci. 2011 Feb;20(2):437-47. doi: 10.1002/pro.577.
Nanolipoprotein particles (NLPs) represent a unique nanometer-sized scaffold for supporting membrane proteins (MP). Characterization of their dynamic shape and association with MP in solution remains a challenge. Here, we present a rapid method of analysis by fluorescence correlation spectroscopy (FCS) to characterize bacteriorhodopsin (bR), a membrane protein capable of forming a NLP complex. By selectively labeling individual components of NLPs during cell-free synthesis, FCS enabled us to measure specific NLP diffusion times and infer size information for different NLP species. The resulting bR-loaded NLPs were shown to be dynamically discoidal in solution with a mean diameter of 7.8 nm. The insertion rate of bR in the complex was ∼55% based on a fit model incorporating two separate diffusion properties to best approximate the FCS data. More importantly, based on these data, we infer that membrane protein associated NLPs are thermodynamically constrained as discs in solution, while empty NLPs appear to be less constrained and dynamically spherical.
纳米脂蛋白颗粒 (NLPs) 是一种独特的纳米级支架，可支持膜蛋白 (MP)。它们在溶液中的动态形状及其与 MP 的结合仍然是一个挑战。在这里，我们提出了一种通过荧光相关光谱 (FCS) 进行快速分析的方法，用于表征能够形成 NLP 复合物的细菌视紫红质 (bR)，一种膜蛋白。通过在无细胞合成过程中选择性标记 NLPs 的各个成分，FCS 使我们能够测量特定的 NLP 扩散时间，并推断不同 NLP 物种的大小信息。结果表明，负载 bR 的 NLP 在溶液中呈动态盘状，平均直径为 7.8nm。基于包含两个单独扩散特性的拟合模型，插入 bR 的插入率约为 55%，以最佳拟合 FCS 数据。更重要的是，基于这些数据，我们推断出与膜蛋白相关的 NLP 在溶液中作为圆盘受到热力学限制，而空 NLP 似乎受到的限制较小，并且动态呈球形。
