期刊
PROTEIN SCIENCE
卷 20, 期 2, 页码 437-447出版社
WILEY-BLACKWELL
DOI: 10.1002/pro.577
关键词
apolipoprotein; nanolipoprotein particles; nanodiscs; fluorescence correlation spectroscopy; dynamic light scattering; cell-free expression; co-expression
资金
- University of California
- Life Technologies Corporation
- National Science Foundation, The Center for Biophotonics Science and Technology
- University of California, Davis [PHY 0120999]
- U.S. Department of Energy and Lawrence Livermore National Laboratory [DE-AC52-07NA27344, DE-AC52-07NA27244]
- DOE
Nano lipoprotein 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 similar to 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.
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