Sandbox Reserved 993: Difference between revisions

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[[Image:Screenshot_2015-02-22_22.56.09.png|alt text]]<ref>Auld, D.S., Southhall, N. T., Jadhav, A., Johnson, R. L., Diller, D. J., Simeonov, A., Austin, C. P., and Inglese, J. (2008) "Characteristics of chemical libraries for luciferase inhibitory activity", J. Med. Chem. 51(8):2372-2386. doi:10.1021/jm701302v</ref>

== Structure ==

''Photinus pyralis'' luciferase is composed of 550 residues, resulting in a 62 kDa molecular weight. The protein is divided into two domains (the N-terminal domain and the C-terminal domain) by a wide cleft. The N-terminal domain (residues 4-436) is much larger than the C-terminal domain (residues 440-544) and is formed by an antiparallet β-barrel, two β-sheets, and two α-helices. <ref>Conti E., Franks N.P., Brick P. (1996) "Crystal structure of firefly luciferase throws light on a superfamily of adenylate-forming enzymes", Structure 4(3): 287-298.</ref>

''Photinus pyralis'' luciferase is composed of 550 residues, resulting in a 62 kDa molecular weight. The protein is divided into two domains (the N-terminal domain and the C-terminal domain) by a wide cleft. The N-terminal domain (residues 4-436) is much larger than the C-terminal domain (residues 440-544) and is formed by an antiparallet β-barrel, two β-sheets, and two α-helices. <ref>Conti E., Franks N.P., Brick P. (1996) "Crystal structure of firefly luciferase throws light on a superfamily of adenylate-forming enzymes", Structure 4(3): 287-298. doi: 10.1016/S0969-2126(96)00033-0</ref> The secondary structures and motif are arranged to form a five-layered, alternating αβ tertiary structure. The C-terminal domain, on the other hand, is folded into an α+β tertiary structure.

A model for the active site of ''Photinus pyralis'' luciferase was proposed by Branchini and colleagues in 1998 and has held up to more recent data <ref>Zako T., Ayabe K., Aburatani T., Kamiya N., Kitayama A., Ueda H., and Nagamune T. (2003) "Luminescent and substrate binding activities of firefly luciferase N-terminal domain", 1649(2): 183-189. doi: 10.1016/S1570-9639(03)00179-1</ref> In this model, the enzyme contains a binding pocket for ATP as well as a binding pocket for luciferin. The binding pocket for ATP is formed by the residues 316GAP318, 339GYGL342, and V362, and binds to the adenine ring (Branchini et al. 1998). The luciferin binding pocket is comprised of the residues 341GLT343, 346TSA348, 245HHGFGMT251 (helix), 315GGA317 (loop), and R218 (Branchini et al. 1998). The S314-L319 loop and Q338-A348 region were found to be in different positions when substrates were bound (Branchini et al. 1998). Since the loop blocks both of the binding pockets when in the unbound state, it makes sense that a conformational change in the loop must occur.

== Lab Use ==

Sandbox Reserved 993: Difference between revisions

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@@ Line 13: / Line 13: @@
 [[Image:Screenshot_2015-02-22_22.56.09.png|alt text]]<ref>Auld, D.S., Southhall, N. T., Jadhav, A., Johnson, R. L., Diller, D. J., Simeonov, A., Austin, C. P., and Inglese, J. (2008) "Characteristics of chemical libraries for luciferase inhibitory activity", J. Med. Chem. 51(8):2372-2386. doi:10.1021/jm701302v</ref>
 == Structure ==
-''Photinus pyralis'' luciferase is composed of 550 residues, resulting in a 62 kDa molecular weight. The protein is divided into two domains (the N-terminal domain and the C-terminal domain) by a wide cleft. The N-terminal domain (residues 4-436) is much larger than the C-terminal domain (residues 440-544) and is formed by an antiparallet β-barrel, two β-sheets, and two α-helices. <ref>Conti E., Franks N.P., Brick P. (1996) "Crystal structure of firefly luciferase throws light on a superfamily of adenylate-forming enzymes", Structure 4(3): 287-298.</ref>
+''Photinus pyralis'' luciferase is composed of 550 residues, resulting in a 62 kDa molecular weight. The protein is divided into two domains (the N-terminal domain and the C-terminal domain) by a wide cleft. The N-terminal domain (residues 4-436) is much larger than the C-terminal domain (residues 440-544) and is formed by an antiparallet β-barrel, two β-sheets, and two α-helices. <ref>Conti E., Franks N.P., Brick P. (1996) "Crystal structure of firefly luciferase throws light on a superfamily of adenylate-forming enzymes", Structure 4(3): 287-298. doi: 10.1016/S0969-2126(96)00033-0</ref> The secondary structures and motif are arranged to form a five-layered, alternating αβ tertiary structure. The C-terminal domain, on the other hand, is folded into an α+β tertiary structure.
+A model for the active site of ''Photinus pyralis'' luciferase was proposed by Branchini and colleagues in 1998 and has held up to more recent data <ref>Zako T., Ayabe K., Aburatani T., Kamiya N., Kitayama A., Ueda H., and Nagamune T. (2003) "Luminescent and substrate binding activities of firefly luciferase N-terminal domain",  1649(2): 183-189. doi: 10.1016/S1570-9639(03)00179-1</ref> In this model, the enzyme contains a binding pocket for ATP as well as a binding pocket for luciferin. The binding pocket for ATP is formed by the residues 316GAP318, 339GYGL342, and V362, and binds to the adenine ring (Branchini et al. 1998). The luciferin binding pocket is comprised of the residues 341GLT343, 346TSA348, 245HHGFGMT251 (helix), 315GGA317 (loop), and R218 (Branchini et al. 1998). The S314-L319 loop and Q338-A348 region were found to be in different positions when substrates were bound (Branchini et al. 1998). Since the loop blocks both of the binding pockets when in the unbound state, it makes sense that a conformational change in the loop must occur.
 == Lab Use ==