The long term objective is to understand how physical factors such as electrostatic potentials and reduction of dimensionality help to produce a spatial and temporal flow of information through the calcium/phospholipid second messenger system. The three major protein components of this second messenger system - phospholipase C, protein kinase C (PKC), and its major substrate the MARCKS protein - all contain clusters of basic residues that appear to interact transiently with acidic phospholipids in the cell membrane during activation of this system. The main objective for the next five years is to understand how clusters of basic residues on proteins bind to acidic lipids in membranes by studying these three proteins, peptides that mimic the relevant basic regions of these proteins and simple synthetic peptides. This biophysical project is health related: quantitative falsifiable models have been proposed to explain why the tumor promoting phorbol esters act synergistically with acidic phospholipids to activate PKC, and why the binding of a cluster of basic residues on the MARCKS (and related neuromodulin) protein to acidic phospholipids in the plasma membrane is an integral step in both the PKC phosphorylation of Ser residues within these clusters and the concomitant localized release of calmodulin bound to these clusters. Small synthetic basic peptides will be used to determine how the binding to phospholipid vesicles depends on the number, distribution, and chemical nature of the basic residues, as well as the temperature and pressure. The magnitude of the """"""""entropy price"""""""" paid upon binding also will be measured. the highly oversimplified Gouy- Chapman/mass action model for electrostatic lipid-peptide interactions will be improved by incorporating existing diffraction and 2H-NMR data about the orientation of the polar head groups into a molecular model of a membrane, then assigning realistic partial charges to atoms on these head groups and using the three dimensional Poisson-Boltzmann equation to calculate the electrostatic potential. Structural information about peptides necessary for this new model will be obtained using circular dichroism and 2D NMR in collaboration with D. Cafiso (U. Virginia). The membrane binding of the phosphorylated and unphosphorylated forms of a 25-amino acid peptide that contains the PKC phosphorylation sites and calmodulin binding domain of MARCKS will be studied, as will the binding of this region on the intact protein. Techniques to be used include filtration, equilibrium dialysis, centrifugation of sucrose-loaded vesicles, electrophoretic mobility, calorimetry, monolayer pressure, and fluorescence spectroscopy.
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