Claims
- 1. An isolated lipid binding domain comprising the sequence
- 2. The lipid binding domain of claim 1, wherein u is F, V, I, L, M, or W.
- 3. The lipid binding domain of claim 1, wherein j is R or K.
- 4. The lipid binding domain of claim 1, wherein each x is selected to be the same as a corresponding amino acid in SARA protein.
- 5. The lipid binding domain of claim 1, where the sequence comprises the FYVE sequence of the SARA protein (SEQ ID NO:10).
- 6. The lipid binding domain of claim 1, wherein the domain comprises a synthetic sequence.
- 7. The lipid binding domain of claim 1, where the domain comprises a peptoid sequence.
- 8. The lipid binding domain of claim 1, where the domain comprises a peptidomimetic sequence.
- 9. The lipid binding domain of claim 1, where the domain comprises a Turret loop selected from the group consisting of: AFFR (SEQ ID NO:14), AFIR (SEQ ID NO:15), AIFR (SEQ ID NO:16), AFFK (SEQ ID NO:17), AFIK (SEQ ID NO:18), AIFK (SEQ ID NO:19), and TFTK (SEQ ID NO:20).
- 10. The lipid binding domain of claim 1, wherein u is Y, M, T, or F.
- 11. A lipid binding molecule comprising
(a) a lipid binding domain of claim 1; and (b) a reporter group linked to the lipid binding domain.
- 12. The lipid binding molecule of claim 11, wherein the lipid binding domain binds to phosphatidylinositol 3-phosphate.
- 13. The lipid binding molecule of claim 11, wherein the lipid binding domain binds to phosphatidylinositol 3-phosphate on an endosome.
- 14. The lipid binding molecule of claim 11, wherein the lipid binding domain binds to phosphatidylinositol 3-phosphate on a liposome.
- 15. The lipid binding molecule of claim 11, wherein the reporter group binds to a substrate.
- 16. The lipid binding molecule of claim 11, wherein the reporter group comprises one member of a binding pair, and wherein a substrate comprises a second member of the binding pair.
- 17. The lipid binding molecule of claim 11, wherein the reporter group is selected from the group consisting of glutathione-S-transferase (GST), His X6 (6His), FLAG, Green Fluorescent Protein, chitin binding protein, cellulase, maltose binding protein, dihydrofolate reductases, FK506 binding protein (FKBP), FKBPF36V, an antibody, a fluorescently labeled antibody, and an antibody fragment.
- 18. The lipid binding molecule of claim 11, wherein the reporter group comprises a fluorescent moiety.
- 19. The lipid binding molecule of claim 18, wherein the fluorescent moiety comprises GFP.
- 20. A method for detecting a lipid within a lipid bilayer in a sample, the method comprising:
a) obtaining a lipid binding molecule of claim 11;b) mixing the lipid binding molecule with the sample under conditions that enable the lipid binding molecule to bind to a lipid in the sample to form a complex; and c) detecting the complex as an indication of the presence of the lipid in the sample.
- 21. The method of claim 20, wherein the lipid comprises phosphatidylinositol 3-phosphate.
- 22. The method of claim 20, wherein the lipid binding domain comprises a SARA FYVE domain.
- 23. The method of claim 20, wherein the lipid binding domain comprises naturally occurring amino acids.
- 24. The method of claim 20, wherein the lipid binding domain comprises synthetic amino acids.
- 25. The method of claim 20, wherein the reporter group comprises glutathione S-transferase.
- 26. The method of claim 20, wherein the lipid comprises a cellular lipid membrane.
- 27. The method of claim 26, wherein the cellular lipid membrane comprise an endosome.
- 28. A method of locating a lipid-containing cellular organelle within a cell, the method comprising
a) obtaining a lipid binding molecule of claim 11;b) applying the lipid binding molecule to the cell under conditions that enable the lipid binding molecule to enter the cell; and c) detecting the reporter group of the lipid binding molecule, whereby the location of the reporter group within the cell indicates the location of the cellular organelle.
- 29. The method of claim 28, wherein the method is performed in vivo.
- 30. The method of claim 28, wherein the method is performed in vitro.
- 31. The method of claim 28, wherein the cellular organelle comprises phosphatidylinositol 3-phosphate.
- 32. The method of claim 28, wherein the cellular organelle is an endosome.
- 33. The method of claim 32, wherein the endosome comprises a phagosome.
- 34. The method of claim 28, wherein the cellular organelle is a liposome.
- 35. The method of claim 28, wherein the reporter group provides a visual signal and the method further comprises visualizing the location of the cellular organelle.
- 36. A method of diagnosing a subject for infection by a microorganism, the method comprising
a) obtaining a cell from the subject; b) binding a lipid binding molecule of claim 11 to a phagosome in the cell; c) visualizing the reporter group on a phagosome; and d) determining whether the phagosome is capable of fusing to a lysosome, wherein a phagosome that cannot fuse to a lysosome indicates that the cell is infected by a microorganism.
- 37. The method of claim 36, wherein the microorganism is Mycobacterium tuberculosis.
- 38. The method of claim 36, wherein the cell is derived from a subject at risk for infection by the microorganism.
- 39. The method of claim 36, wherein the subject is a mammal.
- 40. The method of claim 36, wherein the subject is a human.
- 41. A method of determining whether a test compound is a candidate compound for treating Mycobacterium tuberculosis, the method comprising
a) binding a lipid binding molecule of claim 11 to a phagosome in a cell infected by M. tuberculosis; b) applying a test compound to the infected cell; and c) visualizing the phagosome before and after application of the test compound; wherein a phagosome that is capable of fusing to a lysosome after application of the test compound indicates that the test compound is a candidate compound to treat M. tuberculosis.
- 42. The method of claim 41, wherein the infected cell is a cultured cell.
- 43. The method of claim 41, wherein the infected cell is derived from a subject.
- 44. The method of claim 41, wherein the subject is a mammal.
- 45. The method of claim 41, wherein the subject is a human.
- 46. The method of claim 41, wherein the reporter group is visible by microscopy.
- 47. The method of claim 41, wherein the reporter group comprises a fluorescent compound.
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority from U.S. Provisional Patent Application Serial No. 60/400,619, filed on Aug. 2, 2002, the contents of which is incorporated herein by reference in its entirety.
GOVERNMENT FUNDING
[0002] This invention was made from Government support under grant number 1PO1 DK60564-01 awarded by the NIH and NIDDK. The Government has certain rights in the invention.
Provisional Applications (1)
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Number |
Date |
Country |
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60400619 |
Aug 2002 |
US |