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== Structure ==
== Structure ==
[[Image:HGPR40bind2.png|200 px|right|thumb|Figure 1. Second proposed binding site of hGPR40 with surface shown. Substrate would bind in the deep pocket shown between TM3, 4, and 5.]]
Like most G-protein coupled receptors, hGPR40 contains <scene name='72/721541/Top_view_transmembrane_helices/2'>seven transmembrane helices</scene> (<scene name='72/721541/Top_view_transmembrane_helices/1'>top view of TM helices</scene>). To obtain a [https://en.wikipedia.org/wiki/Protein_crystallization crystallized structure] of the protein, four <scene name='72/721541/Stabilizing_mutations/4'>stabilizing mutations</scene> (<scene name='72/721541/L42a/3'>L42A</scene>, <scene name='72/721541/F88a/4'>F88A</scene>, <scene name='72/721541/G103a/3'>G103A</scene>, <scene name='72/721541/Y202f/3'>Y202F</scene>) were made to increase expression levels and thermal stability of the protein. These mutations did not significantly impact the enzyme's binding affinity with a known agonist, TAK-875.<ref name="Srivastava"/> A <scene name='72/721541/Lysozyme_crimson/2'>T4 Lysozyme</scene> (shown in <FONT COLOR="#DC143C">crimson</FONT>) was also added to intracellular loop 3 to aid in the formation of crystals. T4 Lysozyme also had little effect on TAK-875 binding.<ref name="Srivastava"/> For clarity, lysozyme is removed in all further renderings of hGPR40.
Like most G-protein coupled receptors, hGPR40 contains <scene name='72/721541/Top_view_transmembrane_helices/2'>seven transmembrane helices</scene> (<scene name='72/721541/Top_view_transmembrane_helices/1'>top view of TM helices</scene>). To obtain a [https://en.wikipedia.org/wiki/Protein_crystallization crystallized structure] of the protein, four <scene name='72/721541/Stabilizing_mutations/4'>stabilizing mutations</scene> (<scene name='72/721541/L42a/3'>L42A</scene>, <scene name='72/721541/F88a/4'>F88A</scene>, <scene name='72/721541/G103a/3'>G103A</scene>, <scene name='72/721541/Y202f/3'>Y202F</scene>) were made to increase expression levels and thermal stability of the protein. These mutations did not significantly impact the enzyme's binding affinity with a known agonist, TAK-875.<ref name="Srivastava"/> A <scene name='72/721541/Lysozyme_crimson/2'>T4 Lysozyme</scene> (shown in <FONT COLOR="#DC143C">crimson</FONT>) was also added to intracellular loop 3 to aid in the formation of crystals. T4 Lysozyme also had little effect on TAK-875 binding.<ref name="Srivastava"/> For clarity, lysozyme is removed in all further renderings of hGPR40.


=== Binding Sites ===
=== Binding Sites ===
[[Image:HGPR40bind2.png|200 px|right|thumb|Figure 1. Second proposed binding site of hGPR40 with surface shown. Substrate would bind in the deep pocket shown between TM3, 4, and 5.]]
[[Image:HGPR40bind3.png|200 px|right|thumb|Figure 2. Third proposed binding site of hGPR40 with surface shown. Substrate would bind in the pocket below TM7 and above and inbetween TM 1 and 2.]][http://metislabs.com/radioligand-binding-assays Radioligand binding studies] identified multiple [https://en.wikipedia.org/wiki/Binding_site binding sites] in hGPR40.<ref name="Srivastava"/> [https://en.wikipedia.org/wiki/Agonist Full agonists] and [https://en.wikipedia.org/wiki/Partial_agonist partial agonists] were shown to bind in separate sites with positive [http://www.britannica.com/science/cooperativity cooperativity].<ref name="Lin">PMID:22859723</ref> The <scene name='72/721541/Tak_binding_site/4'>binding site for the partial agonist TAK-875</scene> has been identified, but other binding sites were hypothesized. TAK-875 binds between transmembrane helices 3, 4, and 5 and underneath ECL2. By visual inspection, a second possible binding site was proposed between transmembrane helices 3, 4, and 5 on the intracellular side of the transmembrane helices (Figure 1). Also by visual inspection, a third possible binding site was proposed between transmembrane helices 1, 2, and 7 on the extracellular side of hGPR40, close to the TAK-875 binding site (Figure 2).<ref name="Srivastava"/> These binding sites could potentially serve as regulation points for hGPR40. Many proteins are regulated by the binding of inhibitors.
[[Image:HGPR40bind3.png|200 px|right|thumb|Figure 2. Third proposed binding site of hGPR40 with surface shown. Substrate would bind in the pocket below TM7 and above and inbetween TM 1 and 2.]][http://metislabs.com/radioligand-binding-assays Radioligand binding studies] identified multiple [https://en.wikipedia.org/wiki/Binding_site binding sites] in hGPR40.<ref name="Srivastava"/> [https://en.wikipedia.org/wiki/Agonist Full agonists] and [https://en.wikipedia.org/wiki/Partial_agonist partial agonists] were shown to bind in separate sites with positive [http://www.britannica.com/science/cooperativity cooperativity].<ref name="Lin">PMID:22859723</ref> The <scene name='72/721541/Tak_binding_site/4'>binding site for the partial agonist TAK-875</scene> has been identified, but other binding sites were hypothesized. TAK-875 binds between transmembrane helices 3, 4, and 5 and underneath ECL2. By visual inspection, a second possible binding site was proposed between transmembrane helices 3, 4, and 5 on the intracellular side of the transmembrane helices (Figure 1). Also by visual inspection, a third possible binding site was proposed between transmembrane helices 1, 2, and 7 on the extracellular side of hGPR40, close to the TAK-875 binding site (Figure 2).<ref name="Srivastava"/> These binding sites could potentially serve as regulation points for hGPR40. Many proteins are regulated by the binding of inhibitors.


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