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Time-resolved fluorescence anisotropy and fluctuation correlation analysis of major histocompatibility complex class

Ahmed A Heikal1

  • 1Department of Chemistry and Biochemistry, Swenson College of Science and Engineering, University of Minnesota-Duluth, Duluth, MN 55812, USA; Department of Pharmacy Practice and Pharmaceutical Sciences, College of Pharmacy, University of Minnesota-Duluth, Duluth, MN 55812, USA.

Methods (San Diego, Calif.)
|July 2, 2013
PubMed

Insights

Major histocompatibility complex class I (MHC(I)) proteins exhibit distinct structural flexibility and associations within the endoplasmic reticulum (ER). This study reveals real-time dynamics of GFP-labeled MHC(I) alleles in living cells, offering new insights into their intracellular behavior.

Area of Science:

  • Immunology
  • Cell Biology
  • Biophysics

Background:

  • Major histocompatibility complex class I (MHC(I)) proteins are synthesized in the endoplasmic reticulum (ER) and are vital for immune responses.
  • Their orientation and mobility within the cell are critical for presenting peptides to T-cell receptors and triggering cell destruction.
  • Understanding the structural flexibility and intracellular associations of MHC(I) is essential for comprehending immune system function.

Purpose of the Study:

  • To investigate the structural flexibility and intracellular associations of GFP-encoded MHC(I) alleles (H2L(d)) in cultured mouse fibroblast cells.
  • To characterize the real-time dynamics of MHC(I) proteins within the ER using advanced imaging techniques.
  • To provide new insights into the behavior of GFP-labeled MHC(I) alleles in living cells.

Main Methods:

  • Utilizing time-resolved fluorescence anisotropy to measure the rotational dynamics of H2L(d)GFPin and H2L(d)GFPout in the ER.
  • Employing fluorescence correlation spectroscopy (FCS) to determine the diffusion coefficients of MHC(I) constructs at the single-molecule level.
  • Conducting immunoblotting to confirm the molecular weight of the GFP-labeled MHC(I) constructs.

Main Results:

  • Time-resolved fluorescence anisotropy indicated a dominant tumbling motion (56±7 ns) and fast conformational flexibility for H2L(d)GFPin in the ER, contrasting with restricted rotation for H2L(d)GFPout.
  • Fluorescence correlation spectroscopy revealed single-molecule diffusion coefficients of (1.8±0.5)×10(-9) cm(2)/s for H2L(d)GFPin and (2.1±0.6)×10(-9) cm(2)/s for H2L(d)GFPout in the ER.
  • Immunoblotting confirmed the presence of H2L(d)GFP constructs at approximately 75 kDa, distinct from the 29 kDa of free EGFP.

Conclusions:

  • GFP-labeled MHC(I) alleles exhibit distinct dynamic behaviors and structural flexibility within the ER.
  • These real-time dynamics provide crucial information about the intracellular associations and conformational states of MHC(I) proteins.
  • The findings contribute to a deeper understanding of MHC(I) protein function in the context of cellular immunity.

Keywords:
FCSFLIMH2L(d)GFPMHC class ITime-resolved anisotropy

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