Investigation of the MARCKS-PIP(2) interaction through electron paramagnetic resonance spectroscopy and the use of a novel spin-labeled PIP(2)

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 PIP ₂ 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 δ₁ 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 elector 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. Titration of the MARCKS peptide with lipid vesicles containing proxyl-PIP₂ shows a binding interaction of 2.5–3.5 proxyl-PIP₂ molecules per MARCKS peptide. EPR power saturation of the proxyl-PIP₂ when bound to the MARCKS peptide indicates the label position to be slightly deeper than when not bound to MARCKS. Spin-labeled cysteine derivatives of the MARCKS effector domain show no measurable differences in conformation or position within the bilayer when bound to vesicles containing PIP₂ as opposed to PS as the acidic lipid component. This data is consistent with the hypothesis that the MARCKS protein binds strongly to multiple molecules of PIP₂ in membrane bilayers and regulates their accessibility through non-specific electrostatic interactions.