Measuring rotational diffusion of MHC class I on live cells by polarized FPR

David R. Fooksman, Michael Edidin, B. George Barisas

Research output: Contribution to journalArticle

12 Citations (Scopus)

Abstract

Clustering of membrane proteins is a dynamic process which can regulate cellular function and signaling. The size of receptor and other membrane protein clusters can in principle be measured in terms of their rotational diffusion. However, in practice, measuring rotation of membrane proteins of live cells has been difficult, largely because of the difficulty of rigidly attaching reporter groups to the molecules of interest. Here we show that polarized photobleaching recovery can detect rotation of membrane proteins genetically tagged with yellow fluorescent protein, YFP. MHC class I molecules were engineered with a rigid, in-sequence, YFP tag followed at the C-terminus by a pair of crosslinkable domains. When crosslinker was added we could detect changes in rotational anisotropy decay consistent with clustering of the MHC molecules. This result points the way to use of engineered fluorescent fusion proteins to measure rotational diffusion in native cell membranes.

Original languageEnglish (US)
Pages (from-to)10-16
Number of pages7
JournalBiophysical Chemistry
Volume130
Issue number1-2
DOIs
StatePublished - Oct 2007
Externally publishedYes

Fingerprint

Membrane Proteins
proteins
cells
Molecules
membranes
Cluster Analysis
Photobleaching
Anisotropy
Cell membranes
molecules
Proteins
Fusion reactions
Cell Membrane
Recovery
fusion
recovery
anisotropy
decay

Keywords

  • Anisotropy
  • Clustering
  • FRAP
  • MHC class I
  • pFPR
  • Rotational diffusion

ASJC Scopus subject areas

  • Biochemistry
  • Physical and Theoretical Chemistry
  • Biophysics

Cite this

Measuring rotational diffusion of MHC class I on live cells by polarized FPR. / Fooksman, David R.; Edidin, Michael; Barisas, B. George.

In: Biophysical Chemistry, Vol. 130, No. 1-2, 10.2007, p. 10-16.

Research output: Contribution to journalArticle

Fooksman, David R. ; Edidin, Michael ; Barisas, B. George. / Measuring rotational diffusion of MHC class I on live cells by polarized FPR. In: Biophysical Chemistry. 2007 ; Vol. 130, No. 1-2. pp. 10-16.
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