 |
Laboratory of Molecular Biophysics
Laboratory Journal 2002
Catherine Vénien-Bryan
|
Return to:
Contents.
Catherine Vénien-Bryan
Electron microscope studies
1. Electron crystallography
Structural studies at high resolution are possible using cryo-electron microscopy
and image analysis. Periodic ordering of proteins in two-dimensions
as well as along one-dimensional helices has been used to determine some
important structural features using transmission electron microscopy. Electron
crystallography originally developed by Henderson and Unwin for structure
determination of 2-dimensional (2D) crystals of membrane proteins has now
revealed the atomic resolution of bacteriorhodopsin (Henderson et al.,
1990) , the light harvesting complex (Kühlbrandt et al., 1994)
and the tubulin (Novales et al., 1998).
1.1 Crystallization of soluble protein at the air/water interface
One technique to obtain 2D crystals is the crystallization of soluble protein
on a lipid monolayer first described by Kornberg et al., 1983. This
procedure is based on the formation of 2D crystals of proteins bound to a
ligand-lipid incorporated into a planar lipid layer at the air:water interface.
The classical lipid monolayer technique uses electrostatic interactions
between lipids and proteins. It is however possible to use more specific
interactions. For example, in collaboration with the group of C. Mioskowski
(Paris), we have developed a method of crystallization using the specific
strong interaction between histidine residues and Nickel ion. The polar
head of synthesized lipids carries the Nickel ion whereas a short stretch
of contiguous histidine residues (a His-tag) is located on the C or N terminal
end of the expressed protein
1.1.1 HupR Protein
Karen Davies and Louise Johnson
Using this technique, large, well ordered two-dimensional crystals of the
histidine tagged-HupR protein, a transcriptional regulator from the photosynthetic
bacterium Rhodobacter capsulatus, were obtained (Vénien-Bryan
et al., 1997). HupR (53KDa) is a response regulator of the NtrC subfamily;
it activates the transcription of the structural genes hupSLC, of
[NiFe]hydrogenase.
Many bacterial signalling pathway, particularly those requiring a fast response
to external environmental changes, involve two-component systems. These systems
consist of two proteins: a sensory autophosphorylating protein kinase (histidine
kinase) and a partner response regulator. The response regulators are classified
by the presence of a homologous receiver domain within the protein and can
be subdivided into families based on the number of other functional domains
in the protein. E.g. the CheY family contain only the receiver domain whereas
the OmpR family contain a DNA-binding domain in addition to the receiver
domain.
All response regulator are activated/inactivated by the transfer of a phosphoryl
group from the partner histidine kinase to the aspartate residue in the receiver
domain, but their downstream function depends on the other domains in the
protein. The structures of a number of proteins belonging to the CheY and
OmpR family have been solved to high resolution allowing an incite into the
structural mechanisms underlying the function of these two families of response
regulators.
A third family of response regulators called the NtrC family is less well
known structurally. It contains three functional domains: an N-terminal receiver
domain, a central ATPase domain and a C-terminal DNA-binding domain. The family
is a group of enhance binding proteins that activate the transcription of
enzymes involved in bacterial metabolism e.g. nitrogen fixation, and chemolithotrophic
metabolism. HupR is a member of the NtrC family of response regulators. We
aim to improve the structural understanding of the NtrC family by producing
a medium resolution, 3D reconstruction of HupR using electron microscopy.
A projection map of the full-length protein at 9Å resolution was obtained
by electron cryo-microscopy and image analysis of frozen-hydrated two-dimensional
crystals. The crystals have a p6 plane group with unit cell dimensions
of a=b= 111.6 Å, 
= 120.4o .figure 1 (Vénien-Bryan et al., 2000).
These results provide the first structure at medium resolution of a whole
transcription factor, HupR from the NtrC family. The 3D structure of this
protein is being studied. By tilting the grid within the microscope it is
possible to get different projections views of the protein which are then
combined to produce a medium resolution 3D reconstruction to about 8Å.
|
| Figure 1. A projection map with p6
plane group symmetry ...more |
As well as producing a 3D reconstruction of HupR we also hope to produce
a 3D reconstruction of HupR bound to its enhancer binding site and one of
phosphorylated HupR. These reconstructions will give information about any
conformational change that may occur during the regulatory process of transcription
initiation.
Work is currently being carried out to characterise the binding of the promoter
site to HupR by gel filtration and fluorescence anisotropy. Previous work
by collaborators in Grenoble (Annette Colbeau) suggests that HupR binds to
a palindromic motif of 3 bp with at least 80 bp downstream of this region.
Primary studies show that the palindromic sequence on its own is not sufficient
for binding. Work is in progress to locate the shortest oligonucleotide sequence
sufficient to bind to HupR for use in crystallisation studies.
1.1.2 Other proteins
The recombinant His-tag vascular endothelial cadherin has been crystallised
in 2D on a Ni-lipid monolayer. A projection map at 20 Å have been produced.
This work has been done in collaboration with Rana al-Kurdi, Elizabeth Hewat
and Daniel Gulino, IBS Grenoble, France,
1.2 Crystallization of membrane proteins at the air/water interface.
Jens Dietrich
In 1971 it was first shown by Fromherz (Fromherz 1971) that an ordered arrangement
of protein can be generated underneath a lipid monolayer. The crystallization
on lipid layers is an elegant method because it is possible to work with very
dilute protein solutions and still generate a high local concentration of
protein constrained in two dimensions. Nonetheless the proteins retain sufficient
mobility to allow the organization into crystalline two-dimensional arrays
by lateral diffusion.
Application of surface crystallization on lipid monolayers to membrane proteins
is complicated by the tendency of detergents to solubilize monolayers of regular
lipids. To avoid solubilization of the lipid monolayer by the detergent L.
Lebeau and C. Moskowski in Strasbourg developed a new class of partially fluorinated
lipids. These lipids, when spread at the interface display a high resistance
toward solubilization by detergents. As a test case for the crystallization
of a membrane protein using lipid monolayers served the H+-ATPase
from the plant Arabidopsis thaliana which was expressed in yeast with
a C-terminal His-tag (Jahn et. al. 2001). The plasma membrane H+-ATPase
(AHA2) from A. thaliana is a single-subunit integral membrane protein
with a molecular mass of 104 kDa. It belongs to the family of P-type transport
ATPases and shows large conformational changes during the pumping cycle especial
for function and regulation (Kühlbrandt et al. 2002). P-type ATPases
are widely distributed biological energy transducers that convert the free
energy resulting from ATP hydrolysis into an electrochemical ion gradient
across the membrane. AHA2 became the first membrane protein to be crystallized
with the new method using fluorinated lipidic monolayers (Lebeau et al.
2001).
We are currently in the process of applying this technique to other His-tagged
membrane proteins. An ellipsometer has been installed in order to follow
the absorption process of the protein to the lipid monolayer. This non-invasive
method is sensitive to the density and thickness of the interface layer and
therefore especially useful for investigation of surface monolayer behaviour
(Vénien-Bryan et. al. 1998).
1.3 Amphipols - a novel family of surfactants.
In collaboration with JL Popot, IBPC Paris.
Amphipols are a novel family of surfactants (Tribet et. al. 1996).
They are composed of a strongly hydrophilic polymeric backbone which is grafted
with hydrophobic chains, making them amphiphilic. These amphiphilic polymers
bind to hydrophobic surfaces of proteins in a non-covalent, quasi-irreversible
manner. Membrane proteins complexed by amphipols are soluble in the absence
of detergent or free amphipols and are generally more stable than in a detergent
solution. Our objective is to develop the application of amphipols to structural
biology, in particular to membrane protein 2D crystallization.
Electron microscopic images of fluorinated lipid monolayers under which
cytochrome bc1/amphipol complexes had been injected have
yielded evidence for protein adsorption. The next step is to find condition
which promotes the crystalline arrangement of these complexes.
1.4 DNA scaffolding
|
Figure 2. Negatively stained EM image of RuvA
...more
|
Louise Johnson, in collaboration with Jonathan Malo, David Sherratt and
Andrew Turberfield.
The aim of this project is to develop a technique for protein structure
determination using self-assembled DNA templates to form engineered 2D crystals.
A pilot study have been made using RuvA, this DNA binding protein is a component
of the Ruv resolvosome that binds and processes Holliday junction intermediates
in homologous recombination. A synthetic protein/DNA crystal have been
produced and a 2D projection map have been calculated at 23 Å resolution,
(Figure 2). We are hoping to develop this method
of crystallization to any protein soluble or membraneous.
References
Fromherz P. 1971. Electron microscopic studies of lipid protein films. Nature
231:267-268.
Henderson, R., et al.., Model for the structure of bacteriorhodopsin
based on high-resolution electron cryo-microscopy. (1990) J. Mol. Biol.
213, 899-929.
Jahn T, Dietrich J, Andersen B, Leidvik B, Otter C, Briving C, Kühlbrandt
W, Palmgren MG. 2001. Large Scale Expression, Purification and 2D Crystallization
of Recombinant Plant Plasma Membrane H(+)-ATPase. J Mol Biol 309(2):465-476.
Kühlbrandt, W., et al., Atomic model of plant-harvesting complex
by electron crystallography. (1994) Nature, 367, 614-621.
Kühlbrandt W, Zeelen J, Dietrich J. 2002. Structure, mechanism, and
regulation of the neurospora plasma membrane H+-ATPase. Science 297(5587):1692-1696.
Lebeau L, Lach F, Venien-Bryan C, Renault A, Dietrich J, Jahn T, Palmgren
MG, Kühlbrandt W, Mioskowski C. 2001. Two-dimensional crystallization
of a membrane protein on a detergent-resistant lipid monolayer. J Mol Biol
308(4):639-47.
Novales, E et al., Structure of the alpha beta tubulin dimer by electron
crystallography. (1998) Nature 391 199-203.
Tribet C, Audebert R, Popot JL. 1996. Amphipols - Polymers That Keep Membrane
Proteins Soluble in Aqueous Solutions. Proceedings of the National Academy
of Sciences of the United States of America 93(26):15047-15050.
Uzgiris, E. & Kornberg, R. Two-dimensional crystallization technique
for imaging macromolecules with application to antigen-antibody-complement
complexes. (1983). Nature, 301, 125-129.
Vénien-Bryan,C., et al. Structural st udy of the response
regulator HupR from Rhosobacter Capsulatus. Electron microscopy of two-dimensional
crystals on a Nickel-chelating lipid. (1997) J. Mol. Biol., 274, 687-692.
Vénien-Bryan C, Lenne PF, Zakri C, Renault A, Brisson A, Legrand
JF, B. B. 1998. Characterization of the Growth of 2d Protein Crystals on
a Lipid Monolayer by Ellipsometry and Rigidity Measurements Coupled to Electron
Microscopy. Biophysical Journal 74(5):2649-2657
Vénien-Bryan, C. et al. Projection structure of a transcriptional
regulator HupR determined by electron cryo-microscopy. (2000) J. Mol. Biol,
296 863-871.
2. Electron microscope studies: Single particle analysis.
In addition to the well established techniques of electron crystallography
and helical three-dimensional reconstruction which can be applied to periodic
or symmetric structures, new powerful methods for single particle analysis
have been developed in the past two decades. The main difference is the way
averaging is performed. Whereas, in electron crystallography or helical reconstruction,
the information for several hundreds or thousands of particles is averaged
directly in a Fourier transform and the reconstruction of the object is obtained
by inverse Fourier or Fourier-Bessel transformation, single particle analysis
works with a large number of individual images of the object and combines
individual image elements. The advantage of this technique is that it is not
necessary to obtain a highly regular arrangement of the object. The method
has been successfully used to carry out 3-D reconstruction of large symmetric
(e.g. the chaperonin) or asymmetric macromolecular assemblies (e.g. ribosomes).
2.1 Phosphorylase kinase
Ed D. Lowe and Louise Johnson
In collaboration with N. Boisset (Paris) and G. M. Carlson (Kansas).
|
Figure 3. Wire representation of the phosphorylase kinase decorated
with glycogen phosphorylaseb. ...more
|
Phosphorylase kinase integrates signals from hormonal messengers and neuronal
stimuli to produce rapid activation of glycogen phosphorylase and subsequent
degradation of glycogen stores either to provide energy to sustain muscle
contraction or, in the liver, to provide other tissues such as the brain with
glucose. It is one of the most complex kinases comprising (
)4 assembly of subunits with a total molecular weight of 1.3 x 106.
The
and
subunits are regulatory; the
subunit is the catalytic subunit; and the
subunit is identical to calmodulin and confers calcium sentivity.
A 3D structure of the holoenzyme PhK has been produced at medium resolution
by electron microscopy and the random conical tilt method using the set of
programs SPIDER (Franck, 1996). The 222 symmetric structure shows a
butterfly like structure 270Å x 225Å by 160Å in overall
dimensions with two wing-like lobes connected by two oblique bridges. Comparison
of the PhK model with previous immunoelectron microscopy studies has allowed
the identification of the
regulatory subunits at the tips of the lobes and the
regulatory subunits at a position on the lobes closer to the cross-bridges.
Structural studies of PhK alone and of PhK decorated with GPb have revealed
the position of the catalytic
subunit of the phosphorylase kinase to be on the side of the lobes close
to the ends figure 3 (Vénien-Bryan et al., 2002). The PhK/GPb
model provides an explanation for the formation of hybrid GPab intermediates
in the PhK catalysed phosphorylation of GPb, as previously observed by other
authors.
We would like to pursue this structural work of Phk at higher resolution
using cryo electron microscopy.
11.2.2 Other Proteins
Louise Johnson and in collaboration with Lori Passmore and David Barford.
The 3D structure of APC is being studied. In this project we would
like to identify and position the various subunits of the APC using a NTA-Nickel
gold cluster.
References
Franck, J. Three dimensional electron microscopy of macromolecular assemblies.
1996, San Diego: Academic Press
Vénien-Bryan, C., E. M. Lowe, Boisset, N. Traxler, K. W.Johnson,
L. N. Carlson, G. M Three-dimensional structure of phosphorylase kinase at
22 A resolution and its complex with glycogen phosphorylase b (2002). Structure
10(1): 33-41.
Return to:
Contents.
Last updated: 14-MAY-2003 17:03