title: Characterizing diffusion dynamics of a membrane protein associated with nanolipoproteins using fluorescence correlation spectroscopy.
abstract: 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.
authors.affiliation: NSF Center for Biophotonics Science and Technology, School of Medicine, University of California Davis, Sacramento, California 95817, USA.
authors.forename: Tingjuan
authors.initials: T
authors.lastname: Gao
authors.forename: Craig D
authors.initials: CD
authors.lastname: Blanchette
authors.forename: Wei
authors.initials: W
authors.lastname: He
authors.forename: Feliza
authors.initials: F
authors.lastname: Bourguet
authors.forename: Sonny
authors.initials: S
authors.lastname: Ly
authors.forename: Federico
authors.initials: F
authors.lastname: Katzen
authors.forename: Wieslaw A
authors.initials: WA
authors.lastname: Kudlicki
authors.forename: Paul T
authors.initials: PT
authors.lastname: Henderson
authors.forename: Ted A
authors.initials: TA
authors.lastname: Laurence
authors.forename: Thomas
authors.initials: T
authors.lastname: Huser
authors.forename: Matthew A
authors.initials: MA
authors.lastname: Coleman
mesh_terms: D001436:Bacteriorhodopsins / Q000737:chemistry* / Q000378:metabolism; D002474:Cell-Free System; D004058:Diffusion; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli; D008027:Light; D016014:Linear Models; D008074:Lipoproteins / Q000737:chemistry*; D018625:Microscopy, Atomic Force; D008967:Molecular Biology; D053758:Nanoparticles / Q000737:chemistry*; D036103:Nanotechnology; D010316:Particle Size; D015202:Protein Engineering; D012542:Scattering, Radiation; D013050:Spectrometry, Fluorescence; D013816:Thermodynamics
chemical_list: D008074:Lipoproteins; D001436:Bacteriorhodopsins