Stuart McLaughlin - US grants

DESCRIPTION: The long term objective is to understand how physical factors such as electrostatics and reduction of dimensionality produce a flow of information through the calcium/phospholipid second messenger system. This proposal focuses on MARCKS, a ubiquitous major substrate of protein kinase C (PKC) that binds reversibly to calmodulin, actin, and membranes. Two hypotheses will be tested. The first hypothesis is that membrane binding of MARCKS requires both insertion of its N-terminal myristate into the interior of the bilayer and interaction of its cluster of basic residues with acidic lipids. PKC phosphorylation of serine residues weakens the electrostatic interaction and produces translocation of MARCKS from membrane to cytoplasm. The second hypothesis is that MARCKS can spontaneously self assemble into lateral domains with monovalent acidic lipids; that other, less prevalent components of this second messenger system (e.g. PKC, Src, and PIP2) will join the domains because of electrostatic considerations; and that these signal transduction domains have important physiological functions. The three specific aims are to test these hypotheses by determining how MARCKS binds to membranes, how it forms domains with acidic lipids, and the significance of these domains. Theoretical calculations using atomic models of membranes and peptides will be combined with experimental measurements to achieve these aims. Measurements of activity, fluorescence, and membrane binding will be performed using a reconstituted signal transduction system composed of phospholipid vesicles and purified proteins (heterotrimeric G protein subunits, phospholipase C, PKC, Src, and MARCKS). MARCKS is medically important because it has been implicated in secretion, cell motility, regulation of the cell cycle and transformation. The studies also are relevant to other medically important proteins that use clusters of basic residues to bind to acidic phospholipids, e.g. K-Ras 4B, Src, and HIV-1 matrix protein. Finally, preliminary data suggest that domains formed by MARCKS and the acidic lipid PIP2 produce a novel amplification of signal transduction based on the concept of positive feedback leading to an "all or none" response.

The objective of this project is to examine the electrostatic potentials on the production of second messengers. The hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2), a lipid located on the cytoplasmic surface of cell membranes, produces inositol 1,4,5-triphosphate (IP3) and diacylglycerol (DG). IP3 induces the release of calcium; DG activates a protein kinase C. Dr. McLaughlin will carry out a number of projects including: 1) studying the binding of polyvalent cations like neomycin to PIP2; 2) labelling the hydrocarbon tails of PIP2 and making fluorescence energy transfer measurements; 3) studying the effect of electrostatic potentials on the ability of calcium to activate a polyphosphoinositide-specific phospholipase C; and 4) continuing theoretical and experimental studies of the role of surface potentials and cooperativity in the binding of polyvalent cations to monovalent anionic lipids in bilayer membranes. Although very few biophysicists are working in this area and little information is available about the physical factors that influence the production and mechanism of action of these second messengers, there is a great deal of evidence present for believing that one of these factors is the electrostatic potential produced by charged lipids such as phosphatidylserine.

9419175 McLaughlin The overall objective is to understand the molecular mechanism by which proteins containing myristate, a 14 carbon acyl chain, interact with biological membranes. The proposal focuses on Src (the product of the v-src oncogene, v-Src, and its cellular homolog, c-Src, are known collectively as Src); the working hypothesis is that membrane binding requires both the hydrophobic insertion of the NH2-terminal myristate into the bilayer and the electrostatic interaction of a cluster of adjacent basic residues with acidic lipids. There are four specific projects. 1) Developing a simple model that describes the synergistic effect of myristate and basic residues on the binding of Src to phospholipid membranes and testing this model by measuring the membrane binding of myristoylated and nonmyristoylated peptides corresponding to the NH2-terminal region of Src. 2) Confirming that the basic residues in the NH2-terminal region of Src are responsible for the electrostatic component of protein-membrane interaction by measuring the membrane binding of native v-Src and c-Src as well as mutant and chimeric versions of these proteins. 3) Measuring the effect of phosphorylation by C and A kinases on the membrane binding of both Src and corresponding peptides. 4) Constructing a molecular model of a phospholipid bilayer and the NH2-terminal region of Src, then using the nonlinear Poisson-Boltzmann equation to calculate theoretically the electrostatic component of the binding. The project is significant because membrane binding is crucial to the function of Src. The biophysical principles that emerge should be applicable to some other myristoylated (e.g. MARCKS) and farnesylated (e.g. K-Ras) proteins. %%% The overall objective is to understand the molecular mechanism by which proteins containing myristate, a 14 carbon acyl chain, interact with biological membranes. The proposal focuses on Src (the product of the v-src oncogene, v-Src, which functions to transfor m cells, and its cellular homolog, c-Src, which is involved in control of the cell cycle, are known collectively as Src); the working hypothesis is that membrane binding requires both the hydrophobic insertion of the NH2-terminal myristate into the bilayer and the electrostatic interaction of a cluster of adjacent basic residues with acidic lipids. These are four specific projects. 1) Developing a simple model that describes the synergistic effect of myristate and basic residues on the binding of Src to phospholipid membranes and testing this model by measuring the membrane binding of synthetic peptides. 2) Confirming that the basic residues in the NH2-terminal region of Src are responsible for the electrostatic component of protein-membrane interaction by measuring the membrane binding of native v-Src and c-Src as well as mutant and chimeric versions of these proteins. 3) Measuring the effect of phosphorylation by C and A kinases on the membrane binding of both Src and corresponding peptides. 4) Constructing a molecular model of a phospholipid bilayer and the NH2-terminal region of Src, then calculating theoretically the electrostatic component of the binding. The project is significant because membrane binding is crucial to the function of Src and the biophysical principles that emerge should be applicable to other myristoylated proteins. ***

9729538 McLaughlin The working hypothesis is that physical factors (e.g. electrostatics, surface pressure) can lead to the self assembly of specific lipids and proteins into lateral domains in cell membranes and that these domains are important for cell signaling. The specific objectives are to understand (1) the molecular mechanisms by which basic peptides form lateral domains in phospholipid vesicles; (ii) how proteins present at high concentrations in the cell membrane, e.g. caveolin, self assemble into domains; and (iii) how other cell signaling proteins, e.g. Src, and lipids, e.g. phosphatidylinositol 4,5,-bisphosphate (PIP2), are sequestered in these domains. Theory and experiment will be developed interactively to provide fundamental biophysical information about this complicated but important problem in signal transduction. Reconstituted systems comprising components of the calcium/phospholipid second messenger system (e.g. PIP2, PKC, Src, G proteins, phospholipase C) and peptides corresponding to the scaffolding region of caveolin (residues 82-101) will be studied using fluorescence, surface pressure, and other techniques. Theoretical calculations will be performed using realistic molecular models of peptides, proteins, and membranes. Four factors that may contribute to domain formation will be examined: self-aggregation of the proteins or peptides; penetration of the polar head group region by W, F, and Y residues; the entropic long rod effect described by Onsager; and electrostatics. Theories that account for each factor's relative contribution to domain formation will be formulated. Preliminary results and calculations show that even simple basic peptides such Lys5 and the scaffolding domain of caveolin can sequester PIP2 in lateral domains via nonspecific electrostatic interactions, which may provide a molecular explanation for the sequestration of this lipid in caveolae. This biophysics project has direct biological relevance because there is good evidence that biologic al membranes contain several different types of lateral domains that are important in signal transduction. For example, the plasma membrane lateral domains called caveolae contain high concentrations of caveolin and signaling molecules such as heterotrimeric G proteins, Src family kinases, PKC, and PIP2; the latter is an important lipid that is both the source of two second messengers and acts to anchor several proteins to membranes. Much of the PIP2 that is hydrolyzed in response to receptor stimulation is localized in the caveolae, suggesting they may be a primary site of agonist-stimulated PIP2 turnover. ***

9907840



Abstract

This grant is being used for the purchase of a 600 MHz wide bore NMR spectrometer for solid state NMR of biological macromolecules and macromolecular assemblies. Structures to be studied include membrane proteins, sub-cellular assemblies and organelles. Processes to be studied include protein folding, structural analysis of catalysis, biopolymers and other chemical systems. The instrument is being installed in the NMR facility at Stoney Brook that is being created through the new structural biology initiative. The major users will focus on problems of membranes, membrane protein structure and signal transduction, collaborating with research groups involved in the study of these processes at Stony Brook.

The long-term objective is to develop technology and instrumentation for measuring the activity of single enzyme molecules that change the charge of their membrane-bound substrates, e.g. lipases, kinases and phosphatases. Combining microelectrophoresis and laser trap technologies, the field/trap apparatus uses the principle of the Millikan oil drop experiment: a silica bead coated with phospholipid bilayer replaces the oil drop and tightly focused laser beam replaces gravity. When an AC field is applied to the coated bead in a salt solution, the electrophoretic force displaces it from its equilibrium position in the laser trap. The displacement, measured with a fast quadrant diode is a proportional to the number of charged lipids (e.g. phosphatidylinositol, 4,5-biphosphate, IPI2) on the outer leaflet of the bead. When a solution containing enzyme (e.g. phospholipase C, PLC) flows past the bead, the proteins adsorb to the surface and change the charge on the bead (e.g. hydrolyze trivalent PIP2 to form the neutral lipid diacylglycerol). A prototype apparatus has been constructed to demonstrate proof-of- principle and used to study PLC-delta. Specific aim 1 is to construct a new apparatus at Stony Brook that will be capable of detecting hydrolysis of 10-100 PIP2 by a single PLC on a bead, initially containing 10,000 PIP2, with a time resolution of 0.1-1.0 sec. Specific Aim 2 is to expand the field/trap approach by adding fluorescence correlation spectroscopy (fcs) capability, which will enable simultaneous measurement of the fluorescence signal from a single enzyme and its activity. The field/trap approach will be applied to study enzymes of great biological and medical importance: PLC-beta isoforms that produce two second messengers when activated by G proteins; the lipid kinase PI3K, which produces another class of second messengers that have been implicated in cancer; and PTEN, a lipid phosphatase that is a highly mutated clinically important tumor suppressor.