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| Content Provider | World Health Organization (WHO)-Global Index Medicus |
|---|---|
| Author | Heikal, Ahmed A. |
| Description | Author Affiliation: Heikal AA ( Department of Chemistry and Biochemistry, Swenson College of Science and Engineering, University of Minnesota-Duluth, Duluth, MN 55812, USA) |
| Abstract | Major histocompatibility complex class I proteins, MHC(I), are expressed in almost all nucleated cells and synthesized in the endoplasmic reticulum (ER). The orientation and mobility of these complexes are crucial in their biological function in the immune system, i.e., the cytosolic pathogen peptides loading and their presentation to T-cell receptors at the plasma membrane, where cell destruction is triggered. Here, we investigate the structural flexibility and associations of GFP-encoded MHC(I) alleles (H2L(d)), namely H2L(d)GFPin and H2L(d)GFPout, in cultured mouse fibroblast cells. Time-resolved fluorescence anisotropy of H2L(d)GFPin in the ER indicates a dominant overall tumbling motion of 56±7 ns (ER), with a fast conformational flexibility, as compared with a restricted rotation of H2L(d)GFPout. At the single-molecule level, the diffusion coefficient of H2L(d)GFPin and H2L(d)GFPout in the ER is (1.8±0.5)×10(-9) and (2.1±0.6)×10(-9) cm(2)/s, respectively, as revealed by fluorescence correlation spectroscopy. A complementary immunoblotting of H2L(d)GFP constructs, isolated from mouse fibroblast cells, reveals band at 75 kDa as compared with 29 kDa of the free EGFP. These real-time dynamics provide new insights into the structural flexibility and intracellular associations of GFP-labeled MHC(I) alleles (H2L(d)) in living cells. |
| File Format | HTM / HTML |
| ISSN | 10462023 |
| Issue Number | 2 |
| Volume Number | 66 |
| e-ISSN | 10959130 |
| Journal | Methods |
| Language | English |
| Publisher | Elsevier |
| Publisher Date | 2014-03-15 |
| Publisher Place | United States |
| Access Restriction | One Nation One Subscription (ONOS) |
| Subject Keyword | Discipline Life sciences, Biochemistry Histocompatibility Antigens Class I Metabolism Animals Cell Line Diffusion Fibroblasts Fluorescence Polarization Green Fluorescent Proteins Chemistry Mice Microscopy, Fluorescence Protein Conformation Protein Transport Recombinant Fusion Proteins Journal Article Research Support, N.i.h., Extramural Research Support, Non-u.s. Gov't Research Support, U.s. Gov't, Non-p.h.s. |
| Content Type | Text |
| Resource Type | Article |
| Subject | Molecular Biology |
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