Chemistry: molecular biology and microbiology – Carrier-bound or immobilized enzyme or microbial cell;... – Enzyme or microbial cell is immobilized on or in an...
Reexamination Certificate
2000-12-01
2003-12-02
Ceperley, Mary E. (Department: 1641)
Chemistry: molecular biology and microbiology
Carrier-bound or immobilized enzyme or microbial cell;...
Enzyme or microbial cell is immobilized on or in an...
C436S524000, C530S391100
Reexamination Certificate
active
06656712
ABSTRACT:
The present invention relates to a method of attaching and/or crystallizing macromolecules, to the chemical reagents used in the said method, to the products obtained as well as to the applications of the said products in the field of materials and of structural biology, in particular as biosensors or as biomaterials.
The knowledge of the structure of proteins and in particular of their active sites is essential for understanding their mechanism of action. Several methods are available for carrying out such studies: X-rays, NMR, electrocrystalography (2D crystallization).
For carrying out the crystallization proper, the technique of two-dimensional crystallization on a lipid film or monolayer, at the air/water interface (E. E. Ugziris et al., Nature, 1983, 301, 125-129), allows the formation of self-organized systems of biological macromolecules (crystals) and the determination of the structures of these molecules by electron microscopy analysis of the crystals obtained.
This method consists in creating a lipid monolayer at the level of an air/liquid interface, the lipids being selected so as to interact with the proteins, present in the liquid phase, which attach to the lipids and then form an organized network.
The attachment of the proteins to the lipids of the monolayer involves chemical interactions at the level of the polar head of the lipids. These interactions are either aspecific, the lipids possessing charged polar ends, giving rise to crystallization through ionic interactions, or specific. In the latter case, the polar head of the lipids carries ligands exhibiting high affinity with the proteins to be attached.
In particular, it has been possible to show that soluble proteins can two-dimensionally crystallize on lipid films which are charged, or which are functionalized by a ligand for the protein studied (B. J. Jap et al., Ultramicroscopy, 1992, 46, 45-84).
More recently, lipids functionalized by metal complexes such as nickel complexes (E. W. Kubalek et al., J. Struct. Biol., 1994, 113, 117-123) have made it possible to crystallize so-called histidine tagged fusion proteins. These proteins indeed possess, at their N- or C-terminal end, a sequence composed of several histidines. It has been possible to show that the attachment of such proteins to a lipid-nickel was due to a strong interaction between the nickel complex and the poly-histidine sequence (C. Vénien-Brian et al., J. Mol. Biol., 1997, 274, 687-692). Such functionalized lipids have made it possible to obtain crystallization, in particular in the case where an appropriate ligand was not available.
However, the crystallization of proteins on lipid films has the disadvantage of being relatively random and of depending on many factors, which are difficult to control simultaneously:
the ligand carried by the lipids should be sufficiently accessible in order to be able to interact with the proteins. This accessibility depends on the length of the spacer arm between the lipid and the ligand: too short, it gives rise to a penetration of the protein inside the lipid layer; too long, it confers an extremely high degree of freedom on the bound protein and increases the incidence of defects in the crystal;
the lipid monolayer should be sufficiently fluid in order to confer a sufficient lateral and rotational mobility on the bound protein, thus allowing the proteins to organize relative to each other and to develop intermolecular contacts, so as to give rise to the crystal;
another difficulty, inherent to crystallization on a lipid monolayer, relates to the stability of the monolayer; indeed, the stability of the air/liquid interface is difficult to control. In addition, the lipid monolayer should remain stable, not only before the attachment of the proteins, but also after their attachment, in order to allow the spatial organization of the proteins;
for the microscopy study which follows the crystallization step, it is necessary to produce a multitude of planes, because of the planar nature of the structure obtained.
Consequently, the inventors set themselves the aim of providing a method which makes it possible to attach in solution and to optionally induce self-organization of macromolecules which is more suitable for the requirements of practical use than the 2D crystallization methods previously used.
The subject of the present invention is a method for the attachment and/or self-organization of biological macromolecules, characterized in that it essentially comprises the incubation, without stirring, for at least 15 minutes, of a macromolecule in solution with nanotubes of carbon closed at their ends, under suitable temperature and pH conditions.
Nanotubes were discovered in 1991 (S. Ijima,
Nature
1991, 354, 54-56); since then, they have generated a lot of interest, in particular because of their mechanical properties: high mechanical resistance (M. M. J. Treacy et al.,
Nature
1996, 381, pp. 678-680) and electronic properties: conductor or semiconductor property (J. W. G. Wildöer et al.,
Nature
1998, 391, 59-62: T. W. Odom et al.,
Nature,
1998, 391, 62-64).
Several methods of preparing nanotubes have been described, including that by T. W. Ebbesen et al. (
Nature,
1992, 358, 220-222), which makes it possible to obtain a high yield. Methods of purifying nanotubes have also been described (H. Hiura et al.,
Adv. Mater
., 1995, 7, 275-276; J-M Bonard et al.,
Adv. Mater
., 1997, 9, 827-831 and G. S. Duesberg et al.,
Chem. Commun
. 1998, 435-436); these various methods make it possible to obtain the desired quantities of nanotubes. Methods for the chemical functionalization of nanotubes of carbon have also been described (International Application PCT WO97/32571).
Other methods for the chemical functionalization of nanotubes have also been described; there may be mentioned for example TSANG S. C. et al.,
Journal of the Chemical Society, Chemical Communications
, 1995, 17, 1803-1804 and DAVIS J. J. et al.,
Inorganica Chimica Acta
, 1998, 272, 1, 2, 262-266.
However, they involve chemical reactions which either dramatically modify the geometry of the nanotubes (opening of the ends, partial destruction of the outer sheets), or destroy the intrinsic physical properties of the nanotubes and consequently do not allow organization of biological macromolcules such as proteins, on the nanotubes. Nanotubes modified by such destructive methods are not therefore suitable for adsorption and/or self-organization at their outer surface of synthetic products or of biological macromolecules.
Depending on the technique and the conditions used, several structures of nanotubes may be prepared: the nanotubes have in particular so-called multi-wall nanotube structures (MWNT) or single-wall nanotube structures (SWNT) of graphite. They can be completely, partially or not at all oxidized.
Thus, the nanotubes are, from a chemical point of view, polymers composed solely of carbon and which may comprise up to a million atoms. In accordance with the laws of the chemistry of carbon, the atoms of a nanotube are linked via a solid covalent bond and each atom possesses exactly three neighbours. Thus, regardless of its length, a nanotube is obliged to close at its ends, so as not to leave any chemical bond alone there. In general, its diameter is generally between 1 and 30 nm and its length may be up to several micrometers.
From a physical point of view, nanotubes can be defined as carbon crystals extending in a single direction, the repeating unit having the symmetry of a helix (B. I. Yakobson et al., American Scientist, 1997, 85, 324-337).
According to an advantageous embodiment of the said method, the said biological macromolecules are in particular soluble, membrane or transmembrane proteins, enzymes, antibodies, antibody fragments or nucleic acids.
According to another advantageous embodiment of the said method, the said nanotubes of carbon are functionalized by physical adsorption of a chemical reagent of general formula H-E-L, in which:
H represents a hydrophilic group, selected from the positively or negatively charged groups;
Balavoine Fabrice
Miokowski Charles
Richard Cyrille
Schultz Patrick
Ceperley Mary E.
Commissariat a l'Energie Atomique
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