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Intrasequence GFP in Class I MHC Molecules, a Rigid Probe for Fluorescence Anisotropy Measurements of the Membrane Environment

Jonathan V. Rocheleau ^*, Michael Edidin  and David W. Piston ^*

^* Department of Molecular Physiology and Biophysics, Vanderbilt University, Nashville, Tennessee; and Department of Biology, The Johns Hopkins University, Baltimore, Maryland

Correspondence: Address reprint requests to David W. Piston, 702 Light Hall, Dept. of Molecular Physiology and Biophysics, Vanderbilt University, Nashville, TN 37232. Tel.: 615-322-7030; E-mail: Dave.Piston{at}vanderbilt.edu.

Fluorescence anisotropy measurements can elucidate the microenvironment of a membrane protein in terms of its rotational diffusion, interactions, and proximity to other proteins. However, use of this approach requires a fluorescent probe that is rigidly attached to the protein of interest. Here we describe the use of one such probe, a green fluorescent protein (GFP) expressed and rigidly held within the amino acid sequence of a major histocompatibility complex (MHC) class I molecule, H2L^d. We contrast the anisotropy of this GFP-tagged MHC molecule, H2L^dGFPout, with that of an H2L^d that was GFP-tagged at its C-terminus, H2L^dGFPin. Both molecules fold properly, reach the cell surface, and are recognized by specific antibodies and T-cell receptors. We found that polarized fluorescence images of H2L^dGFPout in plasma membrane blebs show intensity variations that depend on the relative orientation of the polarizers and the membrane normal, thus demonstrating that the GFP is oriented with respect to the membrane. These variations were not seen for H2L^dGFPin. Before transport to the membrane surface, MHC class I associates with the transporter associated with antigen processing complex in the endoplasmic reticulum. The intensity-dependent steady-state anisotropy in the ER of H2L^dGFPout was consistent with FRET homotransfer, which indicates that a significant fraction of these molecules were clustered. After MCMV-peptide loading, which supplies antigenic peptide to the MHC class I releasing it from the antigen processing complex, the anisotropy of H2L^dGFPout was independent of intensity, suggesting that the MHC proteins were no longer clustered. These results demonstrate the feasibility and usefulness of a GFP moiety rigidly attached to the protein of interest as a probe for molecular motion and proximity in cell membranes.

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