Characterizing diffusion dynamics of a membrane protein associated with nanolipoproteins using fluorescence correlation spectroscopy

Tingjuan Gao, Craig D. Blanchette, Wei He, Feliza Bourguet, Sonny Ly, Federico Katzen, Wieslaw A. Kudlicki, Paul Henderson, Ted A. Laurence, Thomas R Huser, Matthew A Coleman

Research output: Contribution to journalArticle

17 Citations (Scopus)

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. Published by Wiley-Blackwell.

Original languageEnglish (US)
Pages (from-to)437-447
Number of pages11
JournalProtein Science
Volume20
Issue number2
DOIs
StatePublished - Feb 2011

Fingerprint

Fluorescence Spectrometry
Bacteriorhodopsins
Membrane Proteins
Fluorescence
Spectroscopy
Scaffolds
Labeling
Association reactions

Keywords

  • Apolipoprotein
  • Cell-free expression
  • Co-expression
  • Dynamic light scattering
  • Fluorescence correlation spectroscopy
  • Nanodiscs
  • Nanolipoprotein particles

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology

Cite this

Characterizing diffusion dynamics of a membrane protein associated with nanolipoproteins using fluorescence correlation spectroscopy. / Gao, Tingjuan; Blanchette, Craig D.; He, Wei; Bourguet, Feliza; Ly, Sonny; Katzen, Federico; Kudlicki, Wieslaw A.; Henderson, Paul; Laurence, Ted A.; Huser, Thomas R; Coleman, Matthew A.

In: Protein Science, Vol. 20, No. 2, 02.2011, p. 437-447.

Research output: Contribution to journalArticle

Gao, T, Blanchette, CD, He, W, Bourguet, F, Ly, S, Katzen, F, Kudlicki, WA, Henderson, P, Laurence, TA, Huser, TR & Coleman, MA 2011, 'Characterizing diffusion dynamics of a membrane protein associated with nanolipoproteins using fluorescence correlation spectroscopy', Protein Science, vol. 20, no. 2, pp. 437-447. https://doi.org/10.1002/pro.577
Gao, Tingjuan ; Blanchette, Craig D. ; He, Wei ; Bourguet, Feliza ; Ly, Sonny ; Katzen, Federico ; Kudlicki, Wieslaw A. ; Henderson, Paul ; Laurence, Ted A. ; Huser, Thomas R ; Coleman, Matthew A. / Characterizing diffusion dynamics of a membrane protein associated with nanolipoproteins using fluorescence correlation spectroscopy. In: Protein Science. 2011 ; Vol. 20, No. 2. pp. 437-447.
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