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

• Published in: Biophysical chemistry Vol. 130; no. 1; pp. 10 - 16
• Main Authors: Fooksman, David R., Edidin, Michael, Barisas, B. George
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 01.10.2007
• Subjects: Animals; Anisotropy; Bacterial Proteins - analysis; Bacterial Proteins - genetics; Clustering; Fluorescence Polarization - methods; Fluorescence Recovery After Photobleaching - methods; FRAP; Histocompatibility Antigens Class I - analysis; Histocompatibility Antigens Class I - genetics; Luminescent Proteins - analysis; Luminescent Proteins - genetics; Membrane Proteins - analysis; Membrane Proteins - genetics; MHC class I; Mice; pFPR; Rotational diffusion; MHC class I; FRAP; Rotational diffusion; pFPR; Clustering; Anisotropy
• ISSN: 0301-4622; 1873-4200
• DOI: 10.1016/j.bpc.2007.06.013

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.