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Measuring protein‐protein interactions inside living cells using single color fluorescence correlation spectroscopy. Application to human immunodeficiency virus type 1 integrase and LEDGF/p75
Author(s) -
Maertens Goedele,
Vercammen Jo,
Debyser Zeger,
Engelborghs Yves
Publication year - 2005
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fj.04-3373fje
Subject(s) - integrase , fluorescence correlation spectroscopy , fusion protein , wild type , green fluorescent protein , protein–protein interaction , microbiology and biotechnology , chemistry , mutant , western blot , biology , human immunodeficiency virus (hiv) , gene , virology , recombinant dna , biochemistry , organic chemistry , molecule
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).