User:Jaime B. Hutchison/Sandbox 1
From Proteopedia
(New page: <applet load='1URU' size='450' frame='true' align='right' caption='Insert caption here' /> <scene name='Sandbox_Reserved_126/Dimer/1'>homodimer</scene> The dimer contains <scene name='...) |
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<applet load='1URU' size='450' frame='true' align='right' caption='Insert caption here' /> | <applet load='1URU' size='450' frame='true' align='right' caption='Insert caption here' /> | ||
| + | This is the amphiphysin BAR domain from Drosophila melanogaster. | ||
| + | The biologically active form of this protein is proposed to be a | ||
| + | <scene name='Sandbox_Reserved_126/Dimer/1'>homodimer</scene>. | ||
| - | + | We would like to make fluorescent mutants of this protein for microscopy applications. One way to do this is by attaching maleimide dye to Cys amino acids present in the protein. The homodimer has | |
| - | + | <scene name='Sandbox_Reserved_126/Dimer/2'>4 native cysteine (Cys) amino acids</scene>. | |
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| - | <scene name='Sandbox_Reserved_126/Dimer/2'>4 | + | |
(<font color='orange'>'''Cys is orange'''</font>.) | (<font color='orange'>'''Cys is orange'''</font>.) | ||
| - | + | Before designing the mutant we need to check that there are | |
| - | <scene name='Sandbox_Reserved_126/Dimer/4'> | + | <scene name='Sandbox_Reserved_126/Dimer/4'>Cys sites available on the surface</scene> for maleimide attachment. |
| + | Because Cys 82 looks to be accessible and also because the placement of Cys 66 looks like attaching a fluorophore to it may interfere with membrane binding we will mutate | ||
| + | <scene name='User:Jaime_B._Hutchison/Sandbox_1/Dimer/1'>Cys 66</scene>. | ||
| + | This will be done for each monomer, | ||
| + | <scene name='User:Jaime_B._Hutchison/Sandbox_1/Dimer/5'>Cys 66 in chain A and B</scene>. | ||
| + | The mutation will replace the cysteines at residue 66 with | ||
| + | <scene name='User:Jaime_B._Hutchison/Sandbox_1/Dimer/8'>alanines (Ala)</scene>. | ||
| + | (<font color='purple'>'''Ala is purple'''</font>.) | ||
| + | Each of the mutated monomers will now have only one binding site at | ||
| + | <scene name='User:Jaime_B._Hutchison/Sandbox_1/Dimer/9'>Cys 82</scene>. | ||
| + | The final mutated dimer with Cys 66 replaced with Ala and Cys 82 ready for fluorophore binding is shown | ||
| + | <scene name='User:Jaime_B._Hutchison/Sandbox_1/Dimer/10'>here</scene>. | ||
Current revision
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This is the amphiphysin BAR domain from Drosophila melanogaster. The biologically active form of this protein is proposed to be a .
We would like to make fluorescent mutants of this protein for microscopy applications. One way to do this is by attaching maleimide dye to Cys amino acids present in the protein. The homodimer has . (Cys is orange.) Before designing the mutant we need to check that there are for maleimide attachment. Because Cys 82 looks to be accessible and also because the placement of Cys 66 looks like attaching a fluorophore to it may interfere with membrane binding we will mutate . This will be done for each monomer, . The mutation will replace the cysteines at residue 66 with . (Ala is purple.) Each of the mutated monomers will now have only one binding site at . The final mutated dimer with Cys 66 replaced with Ala and Cys 82 ready for fluorophore binding is shown .
