Organic compounds -- part of the class 532-570 series – Organic compounds – Amino nitrogen containing
Reexamination Certificate
1999-01-27
2001-01-09
Kumar, Shailendra (Department: 1621)
Organic compounds -- part of the class 532-570 series
Organic compounds
Amino nitrogen containing
C564S181000, C564S184000, C564S190000, C564S215000, C564S224000, C530S407000, C530S825000, C510S382000, C436S004000
Reexamination Certificate
active
06172262
ABSTRACT:
FIELD OF THE INVENTION
The invention is directed to a genus of amphiphilic detergents which readily solubilize membrane-bound proteins.
DESCRIPTION OF THE PRIOR ART
The currently accepted model for the molecular structure of a cell membrane is referred to as the fluid mosaic model. In this view, the lipid bilayer of a cell membrane acts as a “sea” in which membrane-bound proteins are dispersed. Membrane-bound proteins are those proteins which associate with the lipid bilayer of a cell membrane. Membrane-bound proteins regulate or otherwise play a role in a vast number of cellular functions, including cell-to-cell signalling, mitosis, viral infectivity, etc. Elucidating the structure and function of membrane-bound proteins is therefore critical to a complete understanding of overall cellular functionality.
However, separating a membrane-bound protein from the lipid components of a bilayer without destroying the essential structure and functionality of the protein is often difficult, if not impossible. This, in turn, makes it equally difficult or impossible to determine the structure of the membrane-bound protein by X-ray crystallography because the impure and/or denatured protein cannot be induced to crystallize.
The lipids which form cell membranes are amphiphilic: each lipid molecule contains a polar headgroup and a non-polar region. Likewise, detergents, as a class of compounds, also contain a polar region and a non-polar region. Detergents spontaneously aggregate in aqueous solution into micelles, spherical structures in which the non-polar portions of the detergent molecules point to the center of the structure and the polar regions define the spherical surface of the structure. Detergent micelles are dynamic structures which readily interact with and solubilize hydrophobic material. It is this characteristic which makes detergents invaluable cleaning agents.
Detergent micelles and monomeric detergents can also disrupt lipid bilayers. The disruption of a membrane bilayer by a detergent has been proposed to involve first the insertion of the hydrophobic domain of the detergent into the hydrophobic domain of the membrane bilayer, followed by aggregation of the inserted detergent molecules within the membrane. The regions of aggregated detergent are thought to alter the curvature of the bilayer, causing it to degrade into mixed detergent-lipid aggregates. With sufficient concentration of detergent, the process continues until the membrane bilayer has been completely dissolved and all that remains are mixed detergent-lipid micelles.
This ability to disrupt lipid bilayers makes detergents useful for solubilizing membrane-bound proteins and separating the proteins from the lipid components of the cell membrane. However, crystallizing a detergent-solubilized membrane protein into a structure of sufficient regularity to enable high-resolution X-ray crystallography remains extremely problematic. Despite much effort, well-ordered membrane protein crystals are very difficult to obtain using conventional detergents.
To the inventors' knowledge, only three accounts using rigid amphiphile molecules for solubilization of membrane proteins are found in the prior art. In the first example, Schleicher et al. (Schleicher, A.; Franke, R.; Hofmann, K. P.; Finkelmann, H.; Welte, W.
Biochemistry
1987, 26:5908-5916) found that a biphenyl-based detergent could maintain the solubility of rhodopsin that had initially been solubilized with nonanoyl-N-methylglucamide. The authors suggest that the rigid structure of the detergent provides a hydrophobic core similar to that provided by a lipid bilayer. However, the authors never suggest that the rigidity of the detergent could allow for more facile crystallization of rhodopsin.
The second example of rigid amphiphiles are peptitergents (see Schafmeister, C. E.; Miercke, L. J. W.; Stroud, R. M.
Science
1993, 262:734-738). Peptitergents are rigid &agr;-helical amphiplhilic peptides that have been proposed as solubilization and crystallization agents. However, only bacteriorhodopsin has proven to have long-term stability (weeks) when solubilized by a peptitergent. There are no reports to date of a membrane protein having been crystallized using a peptitergent.
The third example of rigid amphiphiles is modified bile acids like 3-{(3-cholamidopropyl)dimethylammonio}-2-hydroxy-1-propane sulfonate (CHAPSO). See, for example, Azema, J.; Chebli, C.; Bon, M.; Rico-Lattes, I.; Lattes, A.
J. Carbo. Chem.
1995, 14:805-817.
SUMMARY OF THE INVENTION
The invention is directed to amphiphilic compounds comprising Formula I:
wherein R
1
, R
2
, and R
3
are independently selected from the group consisting of C
2
-C
12
straight or branched alkyl; unsubstituted phenyl, biphenyl, C
3
-C
8
cycloalkyl, and C
3
-C
8
cycloalkenyl; and phenyl, biphenyl, C
3
-C
8
cycloalkyl, and C
3
-C
8
cycloalkenyl substituted with one, two, or three C
1
-C
6
straight or branched alkyl groups; or
R
1
and R
2
combined are selected from the group consisting of C
3
-C
8
cycloalkyl, C
3
-C
8
cycloalkenyl; and C
3
-C
8
cycloalkyl and C
3
-C
8
cycloalkenyl substituted with one, two, or three C
1
-C
6
straight or branched alkyl groups;
one of R
4
or R
5
is selected from the group consisting of C
2
-C
6
-straight or branched alkyl-(dimethyl-N-oxide), alkyl-(dimethylamine), alkyl-(trimethylammonium), alkyl-glucosyl, alkyl-maltosyl, glucosyl, maltosyl, and polyethylene(glycosyl);
the other of R
4
or R
5
is selected from the group consisting of H, C
2
-C
6
straight or branched alkyl or alkenyl, C
2
-C
6
-straight or branched alkyl-(dimethyl-N-oxide); alkyl-(dimethylamine), alkyl-(trimethylammonium), alkyl-glucosyl, alkyl-maltosyl, glucosyl, maltosyl, and polyethylene(glycosyl); and salts thereof.
While not being confined to any particular mechanism, the present inventors hypothesize that the flexibility of conventional detergents is what makes them antithetical to the formation of well-ordered crystals of membrane protein-detergent complexes. When a membrane protein-detergent complex crystallizes, detergent is trapped within the crystal lattice in high concentrations. It is believed that the large, disordered domains of conventional detergent complexed to the membrane protein contribute in large measure to the difficulty of growing membrane protein crystals in the first instance, and also contribute to the disorder of proteins within the lattice when crystals are formed.
The invention is therefore directed to amphiphiles as noted above having limited flexibility within their hydrophobic domains. The limited flexibility of the molecules is believed to enhance their ability to solubilize and crystallize membrane-bound proteins into well-order crystals. In short, it is believed that the detergent molecules described herein are sufficiently rigid to minimize intramolecular disorder, long enough to span the gaps within a lattice formed by protein-protein interactions, and yet still sufficiently flexible to maximize the protein-protein contacts which give rise to the lattice structure in a membrane protein-detergent crystal.
The amphiphiles of the present invention can also be used in any application where conventional detergents are utilized. For instance, the amphiphiles of the present invention can be used to lyse cellular membranes. The amphiphiles of the present invention also form micelles in aqueous solution. They can therefore be used to solubilize hydrophobic compounds for dispersion into aqueous solution. More specifically, the subject amphiphiles are useful for solubilizing membrane proteins.
Further aims, objects and advantages of the invention will appear upon a complete reading of the following Detailed Description and attached claims.
DETAILED DESCRIPTION OF THE INVENTION
Abbreviations and Definitions:
BR=Bacteriorhodopsin.
CMC=Critical Micelle Concentration. The concentration of a detergent in an aqueous solution at which the detergent molecules will self-assemble into micelles. Below the CMC, detergents arc mostly monomeric; above the CMC, mi
Gellman Samuel H.
McQuade D. Tyler
DeWitt Ross & Stevens S.C.
Kumar Shailendra
Wisconsin Alumni Research Foundation
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