Use of EPR Power Saturation to Analyze the Membrane-Docking Geometries of Peripheral Proteins: Applications to C2 Domains

Abstract

Despite the central importance of peripheral membrane proteins to cellular signaling and metabolic pathways, the structures of protein-membrane interfaces remain largely inaccessible to high-resolution structural methods. In recent years a number of laboratories have contributed to the development of an electron paramagnetic resonance (EPR) power saturation approach that utilizes site-directed spin labeling to determine the key geometric parameters of membrane-docked proteins, including their penetration depths and angular orientations relative to the membrane surface. Representative applications to Ca 2+ -activated, membrane-docking C2 domains are described. Keywordselectron paramagnetic resonance; electron spin resonance; membrane proteins; site-directed spin labeling PERSPECTIVES AND OVERVIEWMembrane surfaces play a central regulatory role in cellular signaling pathways. The plasma membrane, for example, is one of the primary decision-making centers of the cell where a diverse array of receptors, channels, and signaling complexes make crucial decisions about which signaling pathways are switched on or off in response to extracellular and intracellular signals. Other critical decisions are made at the surfaces of intracellular reticular, Golgi, nuclear, and vacuolar membranes, where membrane protein complexes regulate vesicular and protein synthesis, assembly, trafficking, and degradation. Thus many, if not most, important cellular processes are regulated in part by signaling events occurring at membrane surfaces.Many of the decisions made at membrane surfaces are initiated or modulated by a ubiquitous class of signaling proteins that transiently dock to the lipid bilayer during specific signaling events (17). These regulated peripheral proteins have two states: an aqueous state for which a high-resolution structure can often be obtained in a straightforward manner, and a lipid-bound state for which structural information is much more difficult to acquire. To date, no high-resolution structure of a peripheral protein bound to a lipid bilayer has yet been described. Yet a structural description of the membrane-bound state is essential for a mechanistic understanding of membrane docking and the subsequent regulation of signaling events. In addition, such structural information will likely facilitate the design of drugs engineered to block membrane docking and thereby inhibit certain signaling pathways.Recent studies have used a combination of site-directed spin labeling and electron paramagnetic resonance (EPR) power saturation methods to define the membrane-docking NIH Public Access

The molecular basis of ceramide-1-phosphate recognition by C2 domains

“…Recently, electron paramagnetic resonance ( 44,54,55 ) and X-ray refl ectivity ( 45 ) studies have elucidated the cPLA 2 ␣ -C2 domain orientation in the presence of POPC lipids; however, the orientation when C1P is present in the membrane is still unknown. We found that starting with the same orientation ( 44 ) as that with only POPC, the domain tilts by ‫ف‬ 10° toward the bilayer over the course of the simulation in the presence of C1P ( Fig.…”

Membrane binding domains

“…A number of creative approaches are now being applied to fill in the gaps. The application of site-specific labeling for fluorescence resonance energy transfer (FRET) and electron paramagnetic resonance (EPR; [23] and see Cafiso chapter) was pioneered in studies of C2 domain:membrane interactions. These techniques now appear to be mature enough to be applied to other membrane-binding domains and perhaps to larger membraneassociated assemblies nucleated by these domains.…”

“…A computational electrostatic model has been developed that appears to explain the observed specificity differences through changes both in interaction energies and in the penalty for desolvating charges near the membrane [42]. Considerable effort has gone into defining the geometry of C2 domain interactions with membranes, making this field a test bed for applying FRET, EPR, x-ray reflectivity, and computational methods ( [23]; Cafiso chapter).…”

Electrostatic and Hydrophobic Interactions Differentially Tune Membrane Binding Kinetics of the C2 Domain of Protein Kinase Cα

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