Measuring protein-protein interactions inside living cells using single color fluorescence correlation spectroscopy. Application to human immunodeficiency virus type 1 integrase and LEDGF/p75.
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).
Maertens
Goedele
G
Laboratory of Biomolecular Dynamics, Katholieke Universiteit Leuven, Leuven, Belgium.
Vercammen
Jo
J
Debyser
Zeger
Z
Engelborghs
Yves
Y
D001665:Binding Sites; D004058:Diffusion; D017353:Gene Deletion; D049452:Green Fluorescent Proteins / Q000235:genetics; D019427:HIV Integrase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D036341:Intercellular Signaling Peptides and Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D034741:RNA, Small Interfering / Q000494:pharmacology; D011993:Recombinant Fusion Proteins; D013050:Spectrometry, Fluorescence / Q000379:methods*; D014162:Transfection
D036341:Intercellular Signaling Peptides and Proteins; D034741:RNA, Small Interfering; D011993:Recombinant Fusion Proteins; C485184:enhanced green fluorescent protein; C119380:lens epithelium-derived growth factor; D049452:Green Fluorescent Proteins; D019427:HIV Integrase