Water soluble G-protein coupled receptor
Described herein are recombinant integral membrane proteins having multiple transmembrane domains that have been engineered to be less hydrophobic, through alteration of the amino acid sequence of the native protein, but retain the ability to bind their natural ligand. The decreased hydrophobicity of the described proteins makes them more water soluble than the native protein, which allows the described proteins to be expressed in bacteria in large quantities and isolated in the absence of membranes, all while retaining the ability to interact with known ligands.
1. A recombinant integral membrane protein derived from a native protein and having seven transmembrane domains, the seven transmembrane domains comprising at least 4 transmembrane domains each having at least 3 amino acid mutations compared to the native protein that decrease the overall hydrophobicity of the recombinant integral membrane protein relative to that of the native protein,
wherein the native protein is a human mu opioid receptor, and
wherein the recombinant integral membrane protein assumes an active conformation when bound to a native ligand.
2. The recombinant integral membrane protein of claim 1 , comprising at least 5 transmembrane domains each having at least 3 amino acid mutations that decrease the overall hydrophobicity of the recombinant integral membrane protein relative to that of the native protein.
3. The recombinant integral membrane protein of claim 1 , wherein said recombinant integral membrane protein is characterized as being a G-protein-coupled receptor (GPCR).
4. The recombinant integral membrane protein of claim 1 , wherein the recombinant integral membrane protein has an amino acid sequence that is at least 95% identical to SEQ ID NO: 1.
5. The recombinant integral membrane protein of claim 1 , wherein the recombinant integral membrane protein is water soluble.
6. The recombinant integral membrane protein of claim 1 , wherein the recombinant integral membrane protein further comprises an epitope tag.
7. The recombinant integral membrane protein of claim 6 , wherein the epitope tag comprises 5 consecutive histidine amino acids.
8. A polynucleotide encoding a recombinant integral membrane protein derived from a native protein and having seven transmembrane domains, comprising 4 transmembrane domains each having at least 3 amino acid mutations compared to the native protein that decrease the overall hydrophobicity of the recombinant integral membrane protein relative to that of the native protein,
wherein the native protein is a human mu opioid receptor, and
wherein the recombinant integral membrane protein assumes an active conformation when bound to a native ligand.
9. The polynucleotide of claim 8 , wherein the polynucleotide resides in a bacterium.
10. The polynucleotide of claim 9 , wherein said bacterium is E. coli.
11. A method of identifying a binding compound for a recombinant integral membrane protein derived from a native protein and having seven transmembrane domains, comprising 4 transmembrane domains each having at least 3 amino acid mutations compared to the native protein that decrease the overall hydrophobicity of the recombinant integral membrane protein relative to that of the native protein, comprising:
contacting said recombinant integral membrane protein with a compound, and
determining the affinity of said recombinant integral membrane protein for said compound,
wherein the native protein is a human mu opioid receptor, and
wherein the recombinant integral membrane protein assumes an active conformation when bound to a native ligand.
12. The method of claim 11 , wherein the recombinant integral membrane protein is attached to a surface.
13. The method of claim 11 , wherein the affinity of the compound for the recombinant integral membrane protein is measured by calorimetry, spectral absorption, time-resolved fluorescence resonance energy transfer, or surface plasmon resonance.
14. The method of claim 11 , further comprising obtaining the recombinant, soluble integral membrane protein by expressing in bacteria a polynucleotide encoding the recombinant integral membrane protein and collecting the recombinant, soluble integral membrane protein.
15. The method of claim 11 , wherein said compound comprises one or more ligands, one or more proteins, or both.
16. The method of claim 15 , wherein the compound comprises one or more ligands, and further comprising contacting the compound with the one or more recombinant integral membrane proteins and assessing the affinities of said recombinant integral membrane proteins for said compound.
17. The method of claim 11 , further comprising assessing the structure of the compound.
18. The method of claim 15 , wherein the compound comprises one or more proteins, and further comprising contacting the compound with the one or more recombinant integral membrane proteins and assessing the affinities of said proteins for said compound.