Measuring protein‐protein interactions inside living cells using single color fluorescence correlation spectroscopy. Application to human immunodeficiency virus type 1 integrase and LEDGF/p75
| Autor: | Yves Engelborghs, Zeger Debyser, Jo Vercammen, Goedele N. Maertens |
|---|---|
| Rok vydání: | 2005 |
| Předmět: |
Recombinant Fusion Proteins
Green Fluorescent Proteins Fluorescence correlation spectroscopy HIV Integrase Transfection Biochemistry Protein–protein interaction Green fluorescent protein Diffusion Genetics Humans RNA Small Interfering Binding site Molecular Biology Binding Sites biology Fusion protein Molecular biology Integrase Spectrometry Fluorescence Cytoplasm biology.protein Biophysics Intercellular Signaling Peptides and Proteins Gene Deletion Nuclear localization sequence HeLa Cells Biotechnology |
| Zdroj: | The FASEB Journal. 19:1039-1041 |
| ISSN: | 1530-6860 0892-6638 |
| Popis: | Recently we described the interaction of human immunodeficiency virus type 1 (HIV-1)1 integrase (IN) with a cellular protein, lens epithelium-derived growth factor/transcription co-activator p75 (LEDGF/p75). We now present the study of the diffusion behavior of the three independent domains of IN and LEDGF/p75 using fluorescence correlation microscopy (FCM). We show that diffusion in the cell of the different enhanced green fluorescent protein (EGFP) fusion proteins is described by two components with different fractions and that the average parameters in the nucleus are comparable with those in the cytoplasm. In addition, we demonstrate that specific interaction between EGFP-fused HIV-1 IN and LEDGF/p75 results in a shift in diffusion coefficient (D). The opposite shift was observed in an IN-deletion mutant that does not exhibit LEDGF/p75 binding or in a LEDGF/p75 knock-down experiment using siRNA. We thus demonstrate that protein-protein interactions can be studied in living cells, using single-color FCM (scFCM). |
| Databáze: | OpenAIRE |
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