Cooperative adsorption of ezrin on PIP2-containing membranes.
By means of the quartz crystal microbalance (QCM) and scanning force microscopy (SFM), the adsorption of ezrin, a member of the ezrin/radixin/moesin protein family, on l-alpha-phosphatidylinositol-4,5-bisphosphate (PIP(2)) containing solid-supported membranes was investigated. An increase in the PIP(2) content in 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) membranes resulted in an increased amount of bound ezrin strongly supporting the crucial role of PIP(2) for ezrin recruitment to membranes. No ezrin adsorption to membranes composed of pure POPC was detected. To characterize the binding process in more detail, the kinetics and reversibility of ezrin adsorption were investigated by the QCM technique, showing that the protein remains partly bound after rinsing with pure buffer, which we suspected to be a result of lateral interactions between the proteins. SFM images revealed the formation of two-dimensional ezrin clusters on PIP(2)-doped POPC membranes. Time-elapsed SFM images show that the growth of protein domains occurs from a few nucleation sites. The QCM data in conjunction with the results obtained by SFM led us to propose that the binding process of ezrin occurs in a positive cooperative manner. When lateral interactions of the proteins on the membrane were taken into account, we were able to simulate the kinetics obtained from time-resolved QCM readouts by employing a model developed by Minton. On the basis of the kinetic analysis, we were also able to reconstruct the adsorption isotherm.
Herrig
Alexander
A
Institut für Organische und Biomolekulare Chemie, Georg-August-Universität, Tammannstrasse 2, 37077 Göttingen, Germany.
Janke
Matthias
M
Austermann
Judith
J
Gerke
Volker
V
Janshoff
Andreas
A
Steinem
Claudia
C
D000327:Adsorption; D003598:Cytoskeletal Proteins / Q000737:chemistry / Q000378:metabolism*; D008563:Membrane Lipids / Q000378:metabolism; D008567:Membranes, Artificial; D018625:Microscopy, Atomic Force / Q000379:methods; D019269:Phosphatidylinositol 4,5-Diphosphate / Q000737:chemistry*; D011485:Protein Binding; D013997:Time Factors; D000099149:Ezrin
D003598:Cytoskeletal Proteins; D008563:Membrane Lipids; D008567:Membranes, Artificial; D019269:Phosphatidylinositol 4,5-Diphosphate; D000099149:Ezrin