Chen, Yi

Shigematsu, Hideki

Nagayama, Kuniaki

Sigworth, Fred J.

Imprenta de El Compostelano (Santiago de Compostela)

Galiciana

El Compostelano : diario independiente: Ano XVI Número 4342 - 1935 xaneiro 8

El Compostelano : diario independiente: Ano XVI Número 4338 - 1935 xaneiro 3

Elsevier - Publisher Connector 

Measuring Membrane Potentials with Cryo-EM? 

Monday, February 27, 2012 395a2006-Pos Board B776
Cryo EM Study of Protein Complexes Bound to Streptavidin Crystal
Layers
Bong-Gyoon Han1, Ross W. Walton1, Amos Song2, Peter Hwu2,
Robert M. Glaeser1.
1LBNL, Berkeley, CA, USA, 2U.C.Berkeley, Berkeley, CA, USA.
Conventional cryo-EM specimens use either continuous or holey, glow-
discharge treated carbon films to prepare samples. As a result, particles are
either immobilized by binding to an unnatural (i.e. carbon-film) surface, or
they collide multiple times (due to rapid diffusion) with the freshly blotted
air-water interface. In either case there is a risk that considerable structural het-
erogeneity may occur, or even that particles may undergo severe denaturation.
Binding biotinylated particles to streptavidin monolayer crystals is a more
structure-friendly way to immobilize particles. We have developed a protocol
for preparing biotinylated protein complexes and tethering them to the surface
of streptavidin monolayer-crystals. Three different multi-protein complexes
have been used to demonstrate the generality of this method. The biotinylated
protein complexes bind to the streptavidin crystal layer with high affinity,
whereas the unbiotinylated control samples do not do so. In addition, the
same technique can be used to produce a uniform distribution of Euler angles.
The thermosomes, known to adopt a preferred orientation with other methods
of cryo-EM specimen preparation, have been used as a test sample. Indeed, the
results confirm that a uniform distribution of Euler angles is obtained for the
biotinylated thermosome. We thus conclude that biotinylation, together with
binding to 2-D crystals of streptavidin, is a better method to prepare protein
complexes for cryo-EM study.
2007-Pos Board B777
Three Dimensional Imaging of Maurer’s Clefts in ‘‘Unroofed’’
Plasmodium Falciparum-Infected Erythrocytes by Transmission Electron
Microscopy
Eri H. Hayakawa1, Fuyuki Tokumasu2, Jiro Usukura3, Takafumi Tsuboi4,
Hiroyuki Matsuoka1, Thomas E. Wellems2.
1Lab of Medical Zoology and Parasitology, Depart of Infection and
Immunity, Jichi Medical University, Shimotsuke, Japan, 2Lab of Malaria and
Vector Research, National Institute of Allergy and Infectious Diseases, NIH,
Bethesda, MD, USA, 3Division of Integrated Project, EcoTopia Science
Institute, Nagoya University, Nagoya, Japan, 4Malaria Research Unit,
Cell-Free Science and Technology Research Center, Ehime University,
Matsuyama, Japan.
Plasmodium falciparum human malaria parasites establish unique membrane
systems in the cytoplasm of their host erythrocytes. These systems include
Maurer’s Clefts (MCs), which are evident as small membranous structures
in host erythrocyte periphery and are thought to support the trafficking of
P. falciparum proteins to the host cell surface. Three-dimensional structures
of MCs and their tethering to the host erythrocyte membrane have been
reported from reconstructions of transmission electron microscopy (TEM)
and fluorescence images (Hanssen et al. Mol. Microbiol. 2008 Feb;67(4):
703–18.). To examine the structure of MCs and their proximal interactions in
host erythrocytes, we have used an unroofing/rip-off method to expose the in-
tracellular contents of parasitized erythrocytes and collect structural informa-
tion by TEM. MCs in the unroofed TEM images range in dimension from
200 nm to 700 nm and have easily-resolved tethers that are 170 nm to 450
nm in length. These tethers are present in networks that connect the MCs to
the skeleton of the host erythrocyte membrane.
2008-Pos Board B778
Measuring Membrane Potentials with Cryo-EM?
Yi Chen1, Hideki Shigematsu1,2, Kuniaki Nagayama2, Fred J. Sigworth1.
1Yale University, Department of Cellular and Molecular Physiology,
New Haven, CT, USA, 2National Institute for Physiological Sciences,
Okazaki, Japan.
In materials science it is well known that electrostatic potentials produce phase-
shifts of electron waves which are detectable in phase-contrast electron micro-
scopy. The question arises, are membrane potentials preserved during rapid
freezing and cryo-EM imaging of liposomes? Imaged with 200 keV electrons,
a 200mVmembranepotential in a 30nm liposome is expected to produce a phase
shift of about 50 mrad, resulting in approx. 10% change in image intensity. (The
maximum phase shift due to the liposome membrane alone is about 200 mrad.)
Calculations showed that phase-plate imaging would be necessary to detect the
signal.We used a JEOL JEM2200FSmicroscope equippedwith a Zernike phase
plate having a cut-on frequency of approximately 1/100 nm. Liposomes with or
without added valinomycin were adhered to a carbon film and then washed
a buffer to create a 350:1 Kþ gradient, either inwardly or outwardly directed.
In liposomes in the 20-30 nm size range we observed a contrast difference rela-tive to the no-valinomycin controls as expected frompositive and negativemem-
brane potentials ~100 mV in size. Thus the diffusion potential set up by a Kþ
gradient appears to remain throughout the freezing and imaging process.
2009-Pos Board B779
Molecular Architecture of OPA1, the Dynamin-Related GTPase Involved
in Mitochondrial Fusion
Jurgen Heymann, Aurora Fontainhas, Shunming Fang, Jenny E. Hinshaw.
NIH-NIDDK-LCBB, Bethesda, MD, USA.
Mitochondria are dynamic organelles that constantly undergo fusion and fission
events. Four dynamin-related GTPases have been shown to facilitate mitochon-
drial dynamics. One of them, OPA1, mediates mitochondrial inner membrane
fusion. Mutations in OPA1 have been linked to severe disease phenotypes char-
acterized by progressive degeneration of the retinal ganglion cells, thus leading
to blindness. OPA1 is a nuclear encoded protein that is inserted into the inner
mitochondrial membrane through an N terminal transmembrane domain. There
it undergoes proteolytic processing yielding a long, membrane-bound (OPA1)
and a short, diffusible form (OPA1s). Fusion of the inner mitochondrial mem-
brane requires both forms of OPA1, and the ability of OPA1 to bind and hydro-
lyze GTP. In previous structural studies, we have shown that recombinant
OPA1s binds to lipid preparations with a composition similar to that of the in-
ner mitochondrial membrane (Ban et al., 2010). Our findings indicated that the
interaction of OPA1s with suitable membranes stimulates oligomerization and
self-assembly. Using cryo-electron microscopy, we have visualized and ana-
lyzed the topological arrangement of OPA1 on these tubules and showed that
the protein wraps around the perimeter of these tubes in a helical fashion.
Interestingly, the protein formed a two-dimensional lattice on the lipid bilayer
indicating that specific intermolecular interactions are at play. We are now
investigating the molecular architecture of such OPA1-lipid assemblies using
biochemical and high-resolution electron microscopy (EM) methods. Here,
we report on strategies employed to generate substrates suitable for EM anal-
ysis and on our current insights into the properties and architecture of OPA1
assemblies.
Reference:
Ban, T., Heymann, J. A., Song, Z., Hinshaw, J. E., Chan, D. C.: OPA1 disease
alleles causing dominant optic atrophy have defects in cardiolipin-stimulated
GTP hydrolysis and membrane tubulation. Hum. Mol. Genet. 2010, 19:
2113-22.Molecular Dynamics I
2010-Pos Board B780
Development of a Force Field Topology Database for Detergents for
Molecular Dynamics Simulations with the Amber Force Fields
Ste´phane Abel1, Franc¸ois-Yves Dupradeau2, Beatrice de Foresta3,
Massimo Marchi1.
1Commissariat a` l’Energie Atomique, DSV/iBiTEC-S/SB2SM/LBMS &
URA CNRS 2096, Saclay, France, 2Laboratoire des glucides, UFR de
Pharmacie & CNRS UMR 6219, Universite´ de Picardie - Jules Verne,
Amiens, France, 3Commissariat a` l’Energie Atomique, DSV/iBiTEC-S/
SB2SM/LPM & URA CNRS 2096, Saclay, France.
Membrane proteins (MPs) are amphiphilic in nature. As such, to be manipu-
lated and studied in aqueous solution and to remain biologically active, MPs
need to be transferred in a suitable membrane-mimicking environment such
as detergent solutions. Unfortunately, there is no systematic way of identifying
the ideal detergent for any given MP, as empirical criterions are often used in
this undertaking. Thus, a better knowledge of the interactions between MPs and
detergents at the molecular level could improve this situation. Following this
approach, our work has focused on the molecular dynamics (MD) simulations
of MPs in detergent solutions. In contrast to others FFs (such as CHARMM or
GROMOS), AMBER does not provide specific parameter/FF library set for
detergents. Consequently, studying MPs by MD with the AMBER FF is not
straightforward. In this context, we have developed using R.E.D. Server a com-
plex FF Topology DataBase (FFTopDB) (i.e. RESP partial charges embedded
in a large set of FF libraries) for an ensemble of ionic and non-ionic detergents
compatible with AMBER FF (available at http://q4md-forcefieldtools.org/).
Key points of the procedure are the definition of elementary "building blocks"
with well-identified conformations as well as the derivation of reproducible
RESP charge values. The FFTopDB contains more than 75 molecular frag-
ments, which can be used to construct ~70 different detergents. The approach
has been validated by performing MD simulations on dodecylphosphocholine
(DPC) micelles with the AMBER99SB parameters and compared to results ob-
tained from CHARMM36 and GROMOS53A6 simulations. We find that this
combination of parameters reproduces accurately the micelles structural and


Yi Chen

Hideki Shigematsu

Kuniaki Nagayama

Fred J. Sigworth

Crossref

Biophysical Journal

Measuring Membrane Potentials with Cryo-EM?

El Compostelano : diario independiente: Ano XVI Número 4627 - 1935 decembro 19

file:///data/remote/core/dit/data/elsevier/pdf/662/aHR0cDovL2FwaS5lbHNldmllci5jb20vY29udGVudC9hcnRpY2xlL3BpaS9zMDAwNjM0OTUxMTAzNTA2NQ==.pdf

Measuring Membrane Potentials with Cryo-EM?

Abstract

Similar works

Full text

Available Versions

Galiciana

Galiciana

Elsevier - Publisher Connector

Elsevier - Publisher Connector

Crossref

Galiciana