Single-molecule fluorescence studies of a PH domain: new insights into the membrane docking reaction.
Proteins containing membrane targeting domains play essential roles in many cellular signaling pathways. However, important features of the membrane-bound state are invisible to bulk methods, thereby hindering mechanistic analysis of membrane targeting reactions. Here we use total internal reflection fluorescence microscopy (TIRFM), combined with single particle tracking, to probe the membrane docking mechanism of a representative pleckstrin homology (PH) domain isolated from the general receptor for phosphoinositides, isoform 1 (GRP1). The findings show three previously undescribed features of GRP1 PH domain docking to membranes containing its rare target lipid, phosphatidylinositol (3,4,5)-trisphosphate [PI(3,4,5)P(3)]. First, analysis of surface diffusion kinetics on supported lipid bilayers shows that in the absence of other anionic lipids, the PI(3,4,5)P(3)-bound protein exhibits the same diffusion constant as a single lipid molecule. Second, the binding of the anionic lipid phosphatidylserine to a previously unidentified secondary binding site slows both diffusion and dissociation kinetics. Third, TIRFM enables direct observation of rare events in which dissociation from the membrane surface is followed by transient diffusion through solution and rapid rebinding to a nearby, membrane-associated target lipid. Overall, this study shows that in vitro single-molecule TIRFM provides a new window into the molecular mechanisms of membrane docking reactions.
Knight
Jefferson D
JD
Molecular Biophysics Program, Department of Chemistry and Biochemistry, University of Colorado, Boulder, Colorado, USA.
Falke
Joseph J
JJ
D002462:Cell Membrane / Q000737:chemistry* / Q000378:metabolism; D004058:Diffusion; D006801:Humans; D007700:Kinetics; D016018:Least-Squares Analysis; D008051:Lipid Bilayers; D008565:Membrane Proteins / Q000737:chemistry*; D008856:Microscopy, Fluorescence; D008956:Models, Chemical; D018129:Phosphatidylinositol Phosphates / Q000737:chemistry* / Q000378:metabolism; D010718:Phosphatidylserines / Q000737:chemistry; D038761:Photobleaching; D011485:Protein Binding; D054730:Protein Interaction Domains and Motifs; D018160:Receptors, Cytoplasmic and Nuclear / Q000737:chemistry* / Q000378:metabolism; D013050:Spectrometry, Fluorescence
D008051:Lipid Bilayers; D008565:Membrane Proteins; D018129:Phosphatidylinositol Phosphates; D010718:Phosphatidylserines; D018160:Receptors, Cytoplasmic and Nuclear; C060974:phosphatidylinositol 3,4,5-triphosphate; C040985:phosphatidylinositol receptors