Single Molecule Kinetics of ENTH Binding to Lipid Membranes

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• 作者：Sharon Rozovsky ; Martin B. Forstner ; Holger Sondermann ; Jay T. Groves
• 刊名：The Journal of Physical Chemistry B
• 出版年：2012
• 出版时间：May 3, 2012
• 年：2012
• 卷：116
• 期：17
• 页码：5122-5131
• 全文大小：356K
• 年卷期：v.116,no.17(May 3, 2012)
• ISSN：1520-5207

文摘

Transient recruitment of proteins to membranes is a fundamental mechanism by which the cell exerts spatial and temporal control over proteins鈥?localization and interactions. Thus, the specificity and the kinetics of peripheral proteins鈥?membrane residence are an attribute of their function. Here, we describe the membrane interactions of the interfacial epsin N-terminal homology (ENTH) domain with its target lipid phosphatidylinositol (4,5)-bisphosphate (PtdIns(4,5)P₂). The direct visualization and quantification of interactions of single ENTH molecules with supported lipid bilayers is achieved using total internal reflection fluorescence microscopy (TIRFM) with a time resolution of 13 ms. This enables the recording of the kinetic behavior of ENTH interacting with membranes with physiologically relevant concentrations of PtdIns(4,5)P₂ despite the low effective binding affinity. Subsequent single fluorophore tracking permits us to build up distributions of residence times and to measure ENTH dissociation rates as a function of membrane composition. Furthermore, due to the high time resolution, we are able to resolve details of the motion of ENTH associated with a simple, homogeneous membrane. In this case ENTH鈥檚 diffusive transport appears to be the result of at least three different diffusion processes.