Our purpose is to understand as exactly as possible the chemical details that characterize the key reactions at the membrane aqueous interface. We will continue to proceed at two levels simultaneously; (1) High resolution crystallographic, biochemical and mutational and studies on systems where diffraction-quality crystals already exist; primarily, phospholipase A2. (2) Development of new crystalline systems to study problems where the stereochemistry of the biological process is poorly understood; for example, the receptor/G protein system. 1. Mechanism of phospholipase A2 (PLA2) action: We will continue to focus on the structure and action of phospholipase A2 (PLA2). As a calcium- requiring enzyme that attacks phospholipids in lamellar and micellar aggregates, PLA2 serves as a paradigm for action at the lipid/water interface. Moreover, the attack on membrane phospholipids stands at the control point of the release of many second messengers in the signal transduction cascade. Having established the canonical stereochemistry of both the catalytic mechanism and productive-mode binding, we will proceed as follows to complete our understanding: (a) We will better define the distribution of bound water and the location of protons involved in catalysis by extending X-ray and neutron crystallography to the 1.5 A diffraction limit of parent enzyme and its transition-state analogue complexes; (b) We will explore biochemically and crystallographically new analogues designed to probe the mechanism of catalysis and specific binding. (c) We will study the biochemistry and crystal structure of site- directed mutational variants designed to test the functional inferences drawn from the crystallographic work. (d) We will establish by low dose EM and electron diffraction the disposition of the PLA2 molecule relative to the face of the lamellar substrate aggregate during productive mode binding. (e) We will intensify our focus on the PLA2 from the inflamed synovial cavity. Our purpose here is to better understand the mechanism of arachidonate release and thereby provide a rational approach to the design of anti-inflammatory agents. 2. Receptor/G proteins. We will initiate crystallographic studies on the mechanism of signal transduction through the receptor/G- protein/phosphodiesterase (or phospholipase C) system. Our main goal is to develop and study crystalline specimens that will reveal the stereochemistry of signal transduction between an activated receptor and its cognate G protein and/or of the inhibitory interaction between a phosphorylated receptor and arrestin.
Showing the most recent 10 out of 29 publications
