Investigation of the MARCKS-PIP2 Interaction through Electron Paramagnetic Resonance Spectroscopy and the Use of a Novel Spin-Labeled PIP2
Abstract
The phospholipid, phosphoinositol 4, 5, bisphosphate (PIP2) has been implicated in a number of diverse cellular functions, which include membrane trafficking, regulation of the actin cytoskeleton, endocytosis and exocytosis. However, the mechanism for regulation of PIP2 within the cell during these functions is unknown. One hypothesis for regulation involves controlling the lateral distribution and accessibility of PIP2 within the membrane, possibly through interactions with proteins or lipid domains. A spin labeled derivative of PIP2 (proxyl-PIP2) was synthesized and characterized through electron paramagnetic resonance (EPR) spectroscopy. The proxyl-PIP2 is soluble in chloroform and incorporates into lipid vesicles with the proxyl label resting at the membrane interface. Upon addition of known PIP2 binding molecules such as neomycin and the PLC delta 1 PH domain, the EPR spectrum of proxyl-PIP2 shows an increased linewidth, indicative of a decrease in label motion. Furthermore, the proxyl-PIP2 shows a 1:1 binding upon titration with neomycin, with the same association documented for native PIP2 affinities. Therefore, the proxyl-PIP2 can be used as a probe to investigate and quantitate PIP2 interactions within the membrane bilayer. This probe can also be used to distinguish lateral lipid domains enriched in PIP2. MARCKS is a 87 kDalton protein that contains a highly basic region called the effector domain. The effector domain of the protein is the site phosphorylated by PKC, it binds to calmodulin and helps associate MARCKS to the lipid membrane through electrostatic and hydrophobic interactions. MARCKS binds strongly to PIP2 and may function in a regulatory role by sequestering this lipid. Upon the addition of a peptide from the MARCKS effector domain the proxyl-PIP2 EPR spectrum exhibits changes in linewidth due to a decrease in motional averaging, as well as spin-spin interactions resulting from the close proximity of several proxyl-PIP2.
Document Details
- Document Type
- Technical Report
- Publication Date
- Jan 01, 2002
- Accession Number
- ADA399016
Entities
People
- Michelle E. Rauch
Organizations
- University of Virginia