Crystallization solutions

Details Crystallization solutions Crystallization solutions

2001-07-03

2003-07-29

Utech, Benjamin L. (Department: 1765)

Compositions

Liquid crystal compositions

C436S004000

Reexamination Certificate

active

06599441

ABSTRACT:

FIELD OF THE INVENTION
The present invention relates to solutions that are useful for crystallizing molecules, especially macromolecules such as proteins.
BACKGROUND OF THE INVENTION
Macromolecular X-ray crystallography is an essential tool in modern drug discovery and molecular biology. Using X-ray crystallographic techniques, the three-dimensional structures of biological macromolecules, such as proteins, nucleic acids, and their various complexes, can be determined at practically atomic-level resolution from X-ray diffraction data.
One of the first and most important steps in the X-ray crystal structure determination of a target macromolecule is to grow large, well-diffracting crystals of the macromolecule. As the techniques for collecting and analyzing X-ray diffraction data have become more rapid and automated, crystal growth has become a rate-limiting step in the structure determination process.
Vapor diffusion is the most widely used technique for crystallization in modem macromolecular X-ray crystallography. In this technique, a small volume of the macromolecule sample is mixed with an approximately equal volume of a crystallization solution. The resulting drop of liquid (containing macromolecule and dilute crystallization solution) is sealed in a chamber with a much larger reservoir volume of the crystallization solution. The drop is kept separate from the reservoir of crystallization solvent either by hanging the drop from a glass cover slip or by sitting the drop on a pedestal above the level of the solvent in the reservoir. Over time, the crystallization drop and the reservoir solutions equilibrate via vapor diffusion of volatile chemical species. Supersaturating concentrations of the macromolecule are achieved, resulting in crystallization of the macromolecule sample in the drop.
Lipidic cubic phase (LCP) is a relatively new technique which has been successful in crystallizing integral membrane proteins. Typically, membrane proteins are very hydrophobic, and tend to aggregate amorphously instead of forming well-ordered three-dimensional crystals. In the LCP technique, the protein samples are mixed with a lipid to form a gel-like emulsion of protein and lipid. The lipid in this emulsion forms a cubic, three-dimensional, lattice in which the hydrophobic membrane proteins can form three-dimensional crystals. To set up LCP crystallizations, a portion of LCP emulsion, containing the protein and lipid, is dispensed into a reservoir of crystallization solution. The LCP emulsion must remain as a distinct, separate, phase from the aqueous crystallization solution during the crystallization experiment. Consequently, crystallization solutions used in LCP experiments should not dissolve the LCP emulsion of protein and lipid.
The process of growing biological macromolecule crystals remains a highly empirical process. Macromolecular crystallization is dependent on a host of experimental parameters, including; pH, temperature, the concentration of salts in the crystallization drop, the concentration of the macromolecule to be crystallized, and the concentration of the precipitating agent (of which there are hundreds). In particular, the choice of solute conditions in which to grow crystals continues to be a matter for empirical determination. Consequently, the ability to rapidly and easily generate many crystallization trials is important in determining the ideal conditions for crystallization. Thus, there is a need for sets of preformulated crystallization solutions that can be used to rapidly and easily generate many crystallization trials. More specifically, there is a need for sets of crystallization solutions that can be used to generate numerous LCP crystallization trials.
SUMMARY OF THE INVENTION
In one aspect, the present invention provides sets of crystallization solutions useful, for example, for crystallizing molecules, such as proteins and other macromolecules. The crystallization solution sets are identified herein as Crystallization Solution Set
1
, Crystallization Solution Set
2
, Crystallization Solution Set
3
, Crystallization Solution Set
4
, and Crystallization Solution Set
5
. The composition of Crystallization Solution Sets
1
-
5
are set forth in Tables 1-5 herein.
Thus, in one aspect, the present invention provides sets of crystallization solutions that each comprise a crystallization solution set selected from the group of crystallization solution sets consisting of Crystallization Solution Set
1
, Crystallization Solution Set
2
, Crystallization Solution Set
3
, Crystallization Solution Set
4
, and Crystallization Solution Set
5
.
In another aspect, the present invention provides kits that each comprise at least one crystallization plate and a set of crystallization solutions comprising a set of crystallization solutions selected from the group of crystallization solutions sets consisting of Crystallization Solution Set
1
, Crystallization Solution Set
2
, Crystallization Solution Set
3
, Crystallization Solution Set
4
, and Crystallization Solution Set
5
. In some embodiments of the kits of the invention, the crystallization solutions of the Crystallization Solution Set(s) are disposed within the reservoirs of the crystallization plate(s). The presently preferred crystallization plates for inclusion in the kits of the invention are disclosed in U.S. patent application Ser. No. 09/150,629 (now U.S. Pat. No. 6,039,804), incorporated herein by reference.
Thus, the present invention provides Crystallization Solution Sets and kits that permit a large number of crystallization conditions to be simultaneously tested in order to identify crystallization conditions under which a molecule, especially a biological macromolecule, such as a protein, can be crystallized. The crystallization solutions of the solution sets of the invention can be used in LCP crystallization experiments because they do not dissolve the LCP emulsion of protein and lipid.


REFERENCES:
patent: 5419278 (1995-05-01), Carter
patent: 6039804 (2000-03-01), Kim et al.
patent: 6267935 (2001-07-01), Hol et al.

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