User:Alexis Neyman/Sandbox 1

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== Structure ==
== Structure ==
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[[Image:2D_RRM_and_RS_SRp20_with_fun_shapes3.png|250 px|right|thumb|Figure 1: SRp20 RRM and RS domains are shown.]] The structure of SRp20 was determined by heteronuclear single quantum coherence ([https://en.wikipedia.org/wiki/Heteronuclear_single_quantum_coherence_spectroscopy HSQC]) NMR. The structure is composed of one RNA recognition motif (RRM) at the N-terminus and one Arg/Ser (AR) domain at the C-terminus where the Ser residues are phosphorylated<ref name="corbo">PMID:23685143</ref>. The RRM of SRp20 demonstrates the β1α1β2β3α2β3 topology seen in other [https://en.wikipedia.org/wiki/RNA_recognition_motif RRMs]. The role of the RRM region is to provide substrate specificity where SRp20 interacts with splicing enhancing sequences in mRNA. There have been no determined 3D structures of the RS domain thus it is unclear what its exact role is. However there have been some speculation that it might be involved in aiding protein-protein interactions in the spliceosome. It contains 164 amino acids, half belonging to the RRM and other half to the RS domain (Figure 1). SRp20 has a molecular weight of 19 kDA<ref name="corbo">PMID:23685143</ref>.
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[[Image:2D_RRM_and_RS_SRp20_with_fun_shapes3.png|250 px|right|thumb|Figure 1: SRp20 RRM and RS domains are shown]] The structure of SRp20 was determined by heteronuclear single quantum coherence ([https://en.wikipedia.org/wiki/Heteronuclear_single_quantum_coherence_spectroscopy HSQC]) NMR. The structure is composed of one RNA recognition motif (RRM) at the N-terminus and one Arg/Ser (AR) domain at the C-terminus where the Ser residues are phosphorylated<ref name="corbo">PMID:23685143</ref>. The RRM of SRp20 demonstrates the β1α1β2β3α2β3 topology seen in other [https://en.wikipedia.org/wiki/RNA_recognition_motif RRMs]. The role of the RRM region is to provide substrate specificity where SRp20 interacts with splicing enhancing sequences in mRNA. There have been no determined 3D structures of the RS domain thus it is unclear what its exact role is. However there have been some speculation that it might be involved in aiding protein-protein interactions in the spliceosome. It contains 164 amino acids, half belonging to the RRM and other half to the RS domain (Figure 1). SRp20 has a molecular weight of 19 kDA<ref name="corbo">PMID:23685143</ref>.
=== Poor Solubility Problem ===
=== Poor Solubility Problem ===
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1H-15N HSQC results showed a large hydrophobic β-sheet on the RRM binding to the RNA with all four bases interacting with one of the four aromatic residues via hydrophobic interactions <ref name="Hargous">PMID:17036044</ref>. [https://en.wikipedia.org/wiki/Beta_hairpin β-hairpin] amino acids are hydrogen bonded to bases on nucleic acid targets <ref name="Clery">PMID:18515081</ref>. This suggests that the β-hairpin plays a role in SRp20 selectivity for specific ligands. The researchers used a smaller peptide chain to reduce the NMR broadening seen with longer peptides (allowing for structure determination), with the consequence of reduced binding affinity. The ligand used was <scene name='78/781963/Looking_at_the_ligand/1'>CAUC</scene>. The conformation of U3 and C4 shows that U3 bulges out while C4 partially stacks over A2. Interactions with the RRM that the researchers saw were that <scene name='78/781963/C1_and_tyr_13/1'>C1 stacks with Y13</scene> in β1 and <scene name='78/781963/A2_phe_50/1'>A2 stacks with F50</scene> in β3. These aromatic side chains form hydrophobic interactions with the ligand when stacked (Figure 3). Also, the residue <scene name='78/781963/C1_a2_phe48/1'>F48 inserts between the sugar rings of C1 and A2</scene>. <<scene name='78/781963/C1_binding_pocket3/1'>scene name='78/781963/C1_binding_pocket/1</scene>C1 is recognized definitively by the RRM</scene>. The C1 amino proton hydrogen bonds with the Leu 80 carbonyl oxygen and the Glu 79 side-chain carbonyl oxygen in SRp20. The C1 N3 hydrogen bonds with the Asn 82 amide. The C1 O2 hydrogen bonds with the Ser 81 hydroxyl group <ref name="Hargous">PMID:17036044</ref>.
1H-15N HSQC results showed a large hydrophobic β-sheet on the RRM binding to the RNA with all four bases interacting with one of the four aromatic residues via hydrophobic interactions <ref name="Hargous">PMID:17036044</ref>. [https://en.wikipedia.org/wiki/Beta_hairpin β-hairpin] amino acids are hydrogen bonded to bases on nucleic acid targets <ref name="Clery">PMID:18515081</ref>. This suggests that the β-hairpin plays a role in SRp20 selectivity for specific ligands. The researchers used a smaller peptide chain to reduce the NMR broadening seen with longer peptides (allowing for structure determination), with the consequence of reduced binding affinity. The ligand used was <scene name='78/781963/Looking_at_the_ligand/1'>CAUC</scene>. The conformation of U3 and C4 shows that U3 bulges out while C4 partially stacks over A2. Interactions with the RRM that the researchers saw were that <scene name='78/781963/C1_and_tyr_13/1'>C1 stacks with Y13</scene> in β1 and <scene name='78/781963/A2_phe_50/1'>A2 stacks with F50</scene> in β3. These aromatic side chains form hydrophobic interactions with the ligand when stacked (Figure 3). Also, the residue <scene name='78/781963/C1_a2_phe48/1'>F48 inserts between the sugar rings of C1 and A2</scene>. <<scene name='78/781963/C1_binding_pocket3/1'>scene name='78/781963/C1_binding_pocket/1</scene>C1 is recognized definitively by the RRM</scene>. The C1 amino proton hydrogen bonds with the Leu 80 carbonyl oxygen and the Glu 79 side-chain carbonyl oxygen in SRp20. The C1 N3 hydrogen bonds with the Asn 82 amide. The C1 O2 hydrogen bonds with the Ser 81 hydroxyl group <ref name="Hargous">PMID:17036044</ref>.
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It was also noted that <scene name='78/781963/A2_syn_conformation/1'>A2</scene> adopts an unusual syn conformation. U3 interacts with <scene name='78/781963/U3_hydrophobic_interactions/2'>Phe 48, Trp 40, Ala 42,</scene> and with the β2-3 loop of the RRM. These residues are all hydrophobic, offering a large hydrophobic surface that helps bind the ligand, as well as prevents the solvent from binding. Additionally, C4 is maintained in its position by a <scene name='78/781963/C4_a2_h_bond/1'>hydrogen bond between C4 amino group and the A2 2’ oxygen</scene> <ref name="Hargous">PMID:17036044</ref>. [[Image:Figure_4_C1_and_A2_interactions_Edited2.png|300 px|left|thumb|Figure 3: C1 and A2 on the RNA ligand interacting with hydrophobic residues (Tyr 13, Phe 50, Phe 48) in the RRM domain of the SRp20 protein. Image created using ''Pymol''.]]
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It was also noted that <scene name='78/781963/A2_syn_conformation/1'>A2</scene> adopts an unusual syn conformation. U3 interacts with <scene name='78/781963/U3_hydrophobic_interactions/2'>Phe 48, Trp 40, Ala 42,</scene> and with the β2-3 loop of the RRM. These residues are all hydrophobic, offering a large hydrophobic surface that helps bind the ligand, as well as prevents the solvent from binding. Additionally, C4 is maintained in its position by a <scene name='78/781963/C4_a2_h_bond/1'>hydrogen bond between C4 amino group and the A2 2’ oxygen</scene> <ref name="Hargous">PMID:17036044</ref>. [[Image:Figure_4_C1_and_A2_interactions_Edited2.png|300 px|left|thumb|Figure 3: C1 and A2 on the RNA ligand interacting with hydrophobic residues (Tyr 13, Phe 50, Phe 48) in the RRM domain of the SRp20 protein. Image created using ''Pymol'']]
=== RRM Stability===
=== RRM Stability===

Revision as of 15:42, 10 April 2018

Biological Structure of SRp20

SRp20 Structure

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Alexis Neyman

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