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.

Binding of Apolipoprotein E Inhibits the Oligomer Growth of Amyloid-β Peptide in Solution as Determined by Fluorescence Cross-correlation Spectroscopy

“…Such measurements will demand additional analysis as A␤ has the potential to interact with the lipid phase alone in such particles. However, previous experience (43,44) in using FCS and photon antibunching to determine lipoprotein particle size and protein stoichiometry will be helpful in this pursuit.…”

Recent advances in nanodisc technology for membrane protein studies (2012–2017)

“…This determined that the BR ND insertion rate was 55%. Atomic force microscopy (AFM) measurements were used to validate this rate of insertion . Overall this study provided some basic characterization of BR ND and established a method to analyze membrane proteins by FCS.…”

“…incorporated fluorescently labeled BR protein into NLPD containing labeled lipids and performed FCS analysis. The diffusion curves for BR‐NLPD were compared to empty NLPD, BR, and truncated apolipoprotein in order to determine the diameter of each species . This determined that the BR ND insertion rate was 55%.…”

Cell-free expression of functional receptor tyrosine kinases

“…Cell-free systems employ the protein translation machinery of model organisms to express solely the protein of interest from an input cDNA 12 , providing a convenient platform for the production of membrane-bound proteins 13 14 15 , mutant proteins and labeled proteins. Moreover, the co-expression of other proteins, or the addition of cofactors or auxiliary proteins to cell-free expression reactions, allow the properties of the expressed protein to be assessed under a variety of conditions 14 15 16 . However, cell-free production of large membrane proteins such as ERBB receptors is usually associated with low translation efficiency, poor protein stability and solubility.…”