Method and composition for crystallizing G protein-coupled receptors
View Patent ↗Certain embodiments provide a method for crystallizing a GPCR. The method may employ a fusion protein comprising: a) a first portion of a G-protein coupled receptor (GPCR), where the first portion comprises the TM1, TM2, TM3, TM4 and TM5 regions of the GPCR; b) a stable, folded protein insertion; and c) a second portion of the GPCR, where the second portion comprises the TM6 and TM7 regions of the GPCR.
1. A method comprising:
(a) combining a fusion protein with lipid, wherein the fusion protein comprises, from N-terminus to C-terminus:
(i) a first portion of a G-protein coupled receptor (GPCR), wherein said first portion comprises TM1, TM2, TM3, TM4 and TM5 regions of said GPCR;
(ii) a domain comprising an amino acid sequence that is at least 80% identical to the amino acid sequence of a soluble, autonomously folding, wild-type protein; and
(iii) a second portion of said GPCR, wherein said second portion comprises TM6 and TM7 regions of said GPCR;
(b) subjecting the product of (a) to lipidic phase crystallization conditions; and
(c) determining whether crystals comprising said fusion protein are produced.
2. The method of claim 1 , wherein said first and second portions of said GPCR comprise the amino acid sequence of a naturally occurring GPCR.
3. The method of claim 1 , wherein said first and second portions of said GPCR comprise the amino acid sequence of a non-naturally occurring GPCR.
4. The method of claim 1 , wherein said first portion or said second portion of said GPCR comprises an affinity tag.
5. The method of claim 1 , wherein said fusion protein is bound to a ligand for said GPCR.
6. The method of claim 1 , wherein said domain of (a)(ii) spaces the C-terminal end of the TM5 region and the N-terminal end of the TM6 region of said GPCR such that the closest alpha carbon atoms at said C-terminal end and said N-terminal end are spaced by a distance in the range of from 6 Å to 16 Å.
7. The method of claim 1 , wherein said domain of (a)(ii) is in the range of from 100 to 200 amino acids in length.
8. The method of claim 1 , wherein said domain (a)(ii) has an amino acid sequence that is at least 80% identical to a glucose oxidase.
9. The method of claim 1 , wherein said domain (a)(ii) has an amino acid sequence that is at least 80% identical to a xylanase.
10. The method of claim 1 , wherein said domain (a)(ii) has an amino acid sequence that is at least 80% identical to a cytochrome b562.
11. The method of claim 1 , wherein step (b) is done using a bicelle crystallization method.
12. The method of claim 1 , wherein step (b) is done using a lipidic cubic phase crystallization method.
13. The method of claim 1 , wherein said method comprises combining said fusion protein with a ligand for said GPCR prior to step (b).
14. The method of claim 13 , wherein said ligand is an antagonist, agonist, partial agonist or inverse agonist of said GPCR.
15. A method comprising:
(a) combining a polypeptide with lipid, wherein said polypeptide comprises, from N-terminus to C-terminus:
(i) a first portion of a G-protein coupled receptor (GPCR), wherein said first portion comprises the amino acid sequence that is N-terminal to the IC3 loop of said GPCR;
(ii) a domain comprising an amino acid sequence that is at least 80% identical to the amino acid sequence of a soluble, autonomously folding, wild-type protein; and
(iii) a second portion of said GPCR, wherein said second portion comprises the amino acid sequence that is C-terminal to the IC3 loop of said GPCR;
(b) subjecting the product of (a) to lipidic phase crystallization conditions; and
(c) determining whether crystals comprising said polypeptide are produced.
16. The method of claim 15 , wherein step (b) is done using a bicelle crystallization method.
17. The method of claim 15 , wherein step (b) is done using a lipidic cubic phase crystallization method.
18. The method of claim 15 , wherein said method comprises combining said polypeptide with a ligand for said GPCR prior to step (b).
19. The method of claim 18 , wherein said ligand is an antagonist, agonist, partial agonist or inverse agonist of said GPCR.
20. The method of claim 15 , wherein said first and second portions of said GPCR comprise the amino acid sequence of a non-naturally occurring GPCR.
21. A method comprising:
(a) combining a G-protein coupled receptor (GPCR) with lipid, wherein said GPCR comprises an IC3 loop comprising an amino acid sequence that is at least 80% identical to the amino acid sequence of a soluble, autonomously folding, wild-type protein;
(b) subjecting said GPCR to lipidic phase crystallization conditions in order to obtain crystals of said fusion protein; and
(c) determining whether crystals comprising said GPCR are produced.
22. The method of claim 21 , wherein step (b) is done using a bicelle crystallization method.
23. The method of claim 21 , wherein step (b) is done using a lipidic cubic phase crystallization method.
24. The method of claim 21 , wherein said method comprises combining said fusion protein with a ligand for said GPCR prior to step (b).
25. The method of claim 24 , wherein said ligand is an antagonist, agonist, partial agonist or inverse agonist of said GPCR.
26. The method of claim 21 , wherein said first and second portions of said GPCR comprise the amino acid sequence of a non-naturally occurring GPCR.