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Laboratory of Molecular Biophysics
Laboratory Journal 2002
Catherine Vénien-Bryan


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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, gamma = 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.
see caption
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

see caption
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).
see caption

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 (alpha beta gamma delta )4 assembly of subunits with a total molecular weight of 1.3 x 106. The alpha and beta subunits are regulatory; the gamma subunit is the catalytic subunit; and the delta 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 alpha regulatory subunits at the tips of the lobes and the beta 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 gamma 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.


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