Sandbox2O14: Difference between revisions
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== Overview == | == Overview == | ||
Through the 2024-2025 Spring Semester at Elizabethtown College, analysis of protein 2O14 was performed to determine its enzymatic function and protein class. Determining the function of unknown proteins is beneficial to help understand biological processes. For 2O14 in particular, this protein was found in ''Bacillus subtilis'' from soil and the gastoinestrial tract of ruminants and humans. Understanding its enzymatic function will pinpoint exactly how this organism interacts with the environment and humans. | |||
2O14 exists as monomeric protein complex. Using SPRITE and Chimera revealed one chain that is very long and does not replicate the amino acid chain. This was further confirmed by looking at the X-ray structure of protein 2O14 and it is stating that there is only one unique protein chain. The chain is 366 residues long and the entire complex has a molecular weight of approximately 41.79kDa. | 2O14 exists as monomeric protein complex. Using SPRITE and Chimera revealed one chain that is very long and does not replicate the amino acid chain. This was further confirmed by looking at the X-ray structure of protein 2O14 and it is stating that there is only one unique protein chain. The chain is 366 residues long and the entire complex has a molecular weight of approximately 41.79kDa. | ||
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After performing all the necessary steps to extract the protein, it was found that most of the protein came out in the first elution, elution 1. Then using UV-Vis, 10µL of protein solution was added to a cuvette with a 10:1 ratio of PNPP and PNPP buffer solution. This showed an instantly yellow solution, so it was believed that protein was in the elution. We did the same test again with elution 2, to confirm that the protein was in elution 1, and had similar results. The group then attempted to adjust the pH to be more like dirt and soil pH and got completely opposite results, where the absorbance of the first two tests was around 0.95 and the pH test was around 0.09. With these completely opposite results, it was unclear if elution 1 had the protein. Figures 4, 5, and 6 show the explained experiments above in order. | After performing all the necessary steps to extract the protein, it was found that most of the protein came out in the first elution, elution 1. Then using UV-Vis, 10µL of protein solution was added to a cuvette with a 10:1 ratio of PNPP and PNPP buffer solution. This showed an instantly yellow solution, so it was believed that protein was in the elution. We did the same test again with elution 2, to confirm that the protein was in elution 1, and had similar results. The group then attempted to adjust the pH to be more like dirt and soil pH and got completely opposite results, where the absorbance of the first two tests was around 0.95 and the pH test was around 0.09. With these completely opposite results, it was unclear if elution 1 had the protein. Figures 4, 5, and 6 show the explained experiments above in order. | ||
== Protein Extraction == | |||
The protein was grown inside of ''E.coli'' with a His-tag for resistance to the antibiotic, Ampicillin. Once the protein was grown, the protein solution was centrifuged and sonicated multiple times to extract the expressed protein. For purification, the protein solution was ran through a Ni-NTA column gathering approximately 5 mL of protein elutions in total. | |||
== Figure 4 == | == Figure 4 == | ||
[[Image: | [[Image:1_30mg1stSMALL.jpg]] | ||
Protein test using 10µL of protein from elution and 30mg of PNPP and 3mL of PNPP solution. Measuring absorbance at 405M. | Protein test using 10µL of protein from elution and 30mg of PNPP and 3mL of PNPP solution. Measuring absorbance at 405M. | ||
== Figure 5 == | == Figure 5 == | ||
[[Image:2_30mg2ndelutSMALL.jpg]] | |||
Protein test using 10µL of protein from elution 2 and 30mg of PNPP and 3mL of PNPP solution. Measuring absorbance at 405M. | Protein test using 10µL of protein from elution 2 and 30mg of PNPP and 3mL of PNPP solution. Measuring absorbance at 405M. | ||
== Figure 6 == | == Figure 6 == | ||
[[Image:3_ph560SMALL.jpg]] | |||
Protein test using 10µL of protein from elution 1 and 30mg of PNPP and 3mL of PNPP solution, changing the pH to about 5.60. Measuring absorbance at 405M. | Protein test using 10µL of protein from elution 1 and 30mg of PNPP and 3mL of PNPP solution, changing the pH to about 5.60. Measuring absorbance at 405M. | ||
== Figure 7 == | == Figure 7 == | ||
[[Image:ProteinGel.jpg]] | |||
Image of SDS-PAGE gel that proves that protein was present in the sample. | Image of SDS-PAGE gel that proves that protein was present in the sample. | ||
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== Figure 8 == | == Figure 8 == | ||
[[Image:4_3mgElut1SMALL.jpg]] | |||
Protein test using 10µL of protein from elution 1 and 3mg of PNPP and 3mL of PNPP solution. Measuring absorbance at 405M. | Protein test using 10µL of protein from elution 1 and 3mg of PNPP and 3mL of PNPP solution. Measuring absorbance at 405M. | ||
== Figure 9 == | == Figure 9 == | ||
[[Image:5_3mg1HClSMALL.jpg]] | |||
Protein test using 10µL of protein from elution 1 and 3mg of PNPP and 3mL of PNPP solution and 1 drop of HCL with a pH of about 10.50. Measuring absorbance at 405M. | Protein test using 10µL of protein from elution 1 and 3mg of PNPP and 3mL of PNPP solution and 1 drop of HCL with a pH of about 10.50. Measuring absorbance at 405M. | ||
== Figure 10 == | == Figure 10 == | ||
[[Image:6_10mg10mLPNPASMALL.png]] | |||
Protein test using 10µL of protein from elution 1 and 10mg of PNPA and 10mL of PNPP solution and 1 drop of HCL with a pH of about 10.50. Measuring absorbance at 405M. | Protein test using 10µL of protein from elution 1 and 10mg of PNPA and 10mL of PNPP solution and 1 drop of HCL with a pH of about 10.50. Measuring absorbance at 405M. | ||
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== Figure 11 == | == Figure 11 == | ||
[[Image:7_15mgPNPPSMALL.png]] | |||
Protein test using 10µL of protein from elution 1 and 15mg of PNPA and 3mL of PNPP solution. Measuring absorbance at 405M. | Protein test using 10µL of protein from elution 1 and 15mg of PNPA and 3mL of PNPP solution. Measuring absorbance at 405M. | ||
== Figure 12 == | == Figure 12 == | ||
[[Image:8_PNPAaddproteinSMALL.jpg]] | |||
Protein test with increasing protein solution of 5µL every minute from elution 1 with 15mg of PNPA and 3mL PNPP solution. The pH of the solution is 8.35 and it was measuring absorbance at 405M. | Protein test with increasing protein solution of 5µL every minute from elution 1 with 15mg of PNPA and 3mL PNPP solution. The pH of the solution is 8.35 and it was measuring absorbance at 405M. | ||
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== Figure 1 == | == Figure 1 == | ||
Lowest Calculated affinity image from SwissDock of -6.133 kcal/mol, with the ligand | [[Image:Firstswissdock.png]] | ||
Lowest Calculated affinity image from SwissDock of -6.133 kcal/mol, with the ligand 4-nitrophenyl alpha-glucoside. | |||
== Figure 2 == | == Figure 2 == | ||
[[Image:OutsideActiveSite.png]] | |||
The second lowest calculated binding affinity value, -5.629 kcal/mol, predicted bonding site. Believed that the protein’s binding site is in the center between the beta sheet group on top and the alpha helices on the bottom with the ligand 2-acetamido-2-deoxy-beta-D-glucopyranose. | The second lowest calculated binding affinity value, -5.629 kcal/mol, predicted bonding site. Believed that the protein’s binding site is in the center between the beta sheet group on top and the alpha helices on the bottom with the ligand 2-acetamido-2-deoxy-beta-D-glucopyranose. | ||
== Figure 3 == | == Figure 3 == | ||
[[Image:Smallotherprotein.png]] | |||
Image shows a similar protein, 1PP4, having a different bonding site on the outside of the protein with a binding affinity of -4.429 kcal/mole with the ligand 2-acetamido-2-deoxy-beta-D-glucopyranose. This is interesting due to the similarities of 2O14 and 1PP4, but emphasizes the differences in their active sites | |||
== References == | == References == | ||
