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Structural studies of apoptosis and ion transport regulatory proteins in membranes.

ABSTRACT: Solid-state NMR spectroscopy is being used to determine the structures of membrane proteins involved in the regulation of apoptosis and ion transport. The Bcl-2 family includes pro- and anti-apoptotic proteins that play a major regulatory role in mitochondrion-dependent apoptosis or programmed cell death. The NMR data obtained for (15)N-labeled anti-apoptotic Bcl-xL in lipid bilayers are consistent with membrane association through insertion of the two central hydrophobic alpha-helices that are also required for channel formation and cytoprotective activity. The FXYD family proteins regulate ion flux across membranes, through interaction with the Na(+), K(+)-ATPase, in tissues that perform fluid and solute transport or that are electrically excitable. We have expressed and purified three FXYD family members, Mat8 (mammary tumor protein), CHIF (channel-inducing factor) and PLM (phospholemman), for structure determination by NMR in lipids. The solid-state NMR spectra of Bcl-2 and FXYD proteins, in uniaxially oriented lipid bilayers, give the first view of their membrane-associated architectures.

SUBMITTER: Franzin CM

PROVIDER: S-EPMC3033199 | biostudies-literature | 2004 Feb

REPOSITORIES: biostudies-literature

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Structural studies of apoptosis and ion transport regulatory proteins in membranes.

Franzin Carla M CM Choi Jungyuen J Zhai Dayong D Reed John C JC Marassi Francesca M FM

Magnetic resonance in chemistry : MRC 20040201 2

Solid-state NMR spectroscopy is being used to determine the structures of membrane proteins involved in the regulation of apoptosis and ion transport. The Bcl-2 family includes pro- and anti-apoptotic proteins that play a major regulatory role in mitochondrion-dependent apoptosis or programmed cell death. The NMR data obtained for (15)N-labeled anti-apoptotic Bcl-xL in lipid bilayers are consistent with membrane association through insertion of the two central hydrophobic alpha-helices that are ...[more]

PMID: 14745797

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Project description:Na(+)/Ca(2+) exchanger (NCX) proteins extrude Ca(2+) from the cell to maintain cellular homeostasis. Since NCX proteins contribute to numerous physiological and pathophysiological events, their pharmacological targeting has been desired for a long time. This intervention remains challenging owing to our poor understanding of the underlying structure-dynamic mechanisms. Recent structural studies have shed light on the structure-function relationships underlying the ion-transport and allosteric regulation of NCX. The crystal structure of an archaeal NCX (NCX_Mj) along with molecular dynamics simulations and ion flux analyses, have assigned the ion binding sites for 3Na(+) and 1Ca(2+), which are being transported in separate steps. In contrast with NCX_Mj, eukaryotic NCXs contain the regulatory Ca(2+)-binding domains, CBD1 and CBD2, which affect the membrane embedded ion-transport domains over a distance of ~80 Å. The Ca(2+)-dependent regulation is ortholog, isoform, and splice-variant dependent to meet physiological requirements, exhibiting either a positive, negative, or no response to regulatory Ca(2+). The crystal structures of the two-domain (CBD12) tandem have revealed a common mechanism involving a Ca(2+)-driven tethering of CBDs in diverse NCX variants. However, dissociation kinetics of occluded Ca(2+) (entrapped at the two-domain interface) depends on the alternative-splicing segment (at CBD2), thereby representing splicing-dependent dynamic coupling of CBDs. The HDX-MS, SAXS, NMR, FRET, equilibrium (45)Ca(2+) binding and stopped-flow techniques provided insights into the dynamic mechanisms of CBDs. Ca(2+) binding to CBD1 results in a population shift, where more constraint conformational states become highly populated without global conformational changes in the alignment of CBDs. This mechanism is common among NCXs. Recent HDX-MS studies have demonstrated that the apo CBD1 and CBD2 are stabilized by interacting with each other, while Ca(2+) binding to CBD1 rigidifies local backbone segments of CBD2, but not of CBD1. The extent and strength of Ca(2+)-dependent rigidification at CBD2 is splice-variant dependent, showing clear correlations with phenotypes of matching NCX variants. Therefore, diverse NCX variants share a common mechanism for the initial decoding of the regulatory signal upon Ca(2+) binding at the interface of CBDs, whereas the allosteric message is shaped by CBD2, the dynamic features of which are dictated by the splicing segment.

Project description:In 2014, it was reported that protons can traverse between aqueous phases separated by nominally pristine monolayer graphene and hexagonal boron nitride (h-BN) films (membranes) under ambient conditions. This intrinsic proton conductivity of the one-atom-thick crystals, with proposed through-plane conduction, challenged the notion that graphene is impermeable to atoms, ions, and molecules. More recent evidence points to a defect-facilitated transport mechanism, analogous to transport through conventional ion-selective membranes based on graphene and h-BN. Herein, local ion-flux imaging is performed on chemical vapor deposition (CVD) graphene|Nafion membranes using an "electrochemical ion (proton) pump cell" mode of scanning electrochemical cell microscopy (SECCM). Targeting regions that are free from visible macroscopic defects (e.g., cracks, holes, etc.) and assessing hundreds to thousands of different sites across the graphene surfaces in a typical experiment, we find that most of the CVD graphene|Nafion membrane is impermeable to proton transport, with transmission typically occurring at ≈20-60 localized sites across a ≈0.003 mm2 area of the membrane (>5000 measurements total). When localized proton transport occurs, it can be a highly dynamic process, with additional transmission sites "opening" and a small number of sites "closing" under an applied electric field on the seconds time scale. Applying a simple equivalent circuit model of ion transport through a cylindrical nanopore, the local transmission sites are estimated to possess dimensions (radii) on the (sub)nanometer scale, implying that rare atomic defects are responsible for proton conductance. Overall, this work reinforces SECCM as a premier tool for the structure-property mapping of microscopically complex (electro)materials, with the local ion-flux mapping configuration introduced herein being widely applicable for functional membrane characterization and beyond, for example in diagnosing the failure mechanisms of protective surface coatings.

Dataset Information

Structural studies of apoptosis and ion transport regulatory proteins in membranes.

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Structural studies of apoptosis and ion transport regulatory proteins in membranes.

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