Sandbox 323: Difference between revisions

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There have been previous studies conducted on the 3DS8 protein concluding that it is an alpha-beta hydrolase and originates from the Lin2722 gene in ''Listeria innocua'', a species of Gram-positive bacteria. Studies included database searches and the measurement of enzymatic activity by UV-Vis colorimetric assays using p-nitrophenyl analogs to form p-nitrophenol.[1]

The αβ-hydrolases found in plants have many functions, including ligand receptors and catalytic activity. They are also adaptable and malleable regarding the fold of the protein secondary structure, which makes them an ideal target for research. [2]

</StructureSection>

== Materials ==

- Buffers: Sodium Phosphate buffer, Cell Lysis Buffer Tris-HCl, 10X SDS-PAGE Buffer, Re-Suspension Buffer, 1X Wash Buffer, 1X Elution Buffer

- Solutions for SDS-Page: Coomassie Blue Stain, and Destain

- Pre-cast SDS-Page Gel

== Experimental and Results ==

The beginning experiments were conducted on molecular docking sites to compare the structure of the 3DS8 active site with known proteins. Using the SPRITE database [2], the 3DS8 protein was matched with a few proteases through right-handed superpositions with RMSD values ranging from 1.20-1.40, indicating relative similarities.

The beginning experiments were conducted on molecular docking sites to compare the structure of the 3DS8 active site with known proteins. Using the SPRITE database [3], the 3DS8 protein was matched with a few proteases through right-handed superpositions with RMSD values ranging from 1.20-1.40, indicating relative similarities.

The left-handed superpositions matched with more trypsins and displayed better RMSD values ranging from 0.93-1.11.

The 3DS8 protein did not have many very specific active site matches with known proteins on the Chimera software [3], however, it was very similar to trypsin,

The 3DS8 protein did not have many very specific active site matches with known proteins on the Chimera software [4], however, it was very similar to trypsin,

alpha-chymotrypsin, and proteinase B. The function of 3DS8 is most likely very similar to those since the active sites have the same amino acids and structures that differ within <4 angstroms.

The Dali database [4] was used to determine the conserved sequences within the 3DS8 protein. The majority of the hits were lipases with Z-scores up to 31.8, meaning the proteins are homologous

The Dali database [5] was used to determine the conserved sequences within the 3DS8 protein. The majority of the hits were lipases with Z-scores up to 31.8, meaning the proteins are homologous

to the 3ds8 structure because they are higher than 20. Some hits had high LALI numbers that indicate matching residues in the structure. The active site residues are G104, S102, H222, D188. Each of

these is conserved in the 4 selected protein matches, which means that the active site is conserved. The 3ds8 active site is conserved in many other proteins with similar functions, many of which

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The BLAST database [5] was utilized to search for similar gene sequences and corresponding residue patterns. Using the FASTA sequence, the 3DS8 sequence is matched with similar proteins in an

The BLAST database [6] was utilized to search for similar gene sequences and corresponding residue patterns. Using the FASTA sequence, the 3DS8 sequence is matched with similar proteins in an

alignment that shows similar positions of matching residues. The superfamily of 3DS8 is an αβ-hydrolase. Function and cellular position are unknown, but it is hydrolase-like and exists in bacteria.

The 3DS8 protein is part of the superfamily of alpha-beta hydrolases, so it most likely has the same function. Many structural and sequential similarities are conserved between 3DS8

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InterPro Scan [6] searched for structure and taxonomy relations. The results have information about the domains and families of the protein. At the bottom, there are biological processes,

InterPro Scan [7] searched for structure and taxonomy relations. The results have information about the domains and families of the protein. At the bottom, there are biological processes,

molecular functions, and cellular components to learn more about the protein and where it originates from. InterPro confirmed that 3DS8 is most likely a hydrolase. Since 3DS8 is part of

the hydrolase superfamily, its structure and function are likely to be that of some sort of hydrolase.

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Fractions E1 through E6 were chosen to be run on an SDS-PAGE gel because they contained the highest amounts of protein. After running for an hour on 100-150 mV, the gel was stained and destained. The bands appeared around 30 kDa and fractions E2-E6 were pure.

Fractions E1 through E6 were chosen to be run on an SDS-PAGE gel because they contained the highest amounts of protein. After running for an hour on 100-150 mV, the gel was stained and destained. The bands appeared around 30 kDa and fractions E2-E6 were pure. The controls include running samples from before and after the column and wash.

The ligand used for testing was p-nitrophenyl ~~aldehyde~~ (PNPA) in pH's of 4, 5, 6, 7, and 8. In each cuvette, 5 µL of protein, 50 µL of ligand, and 1.5 mL buffer (varying pH) were added. Absorbance was measured at 405 nm for 30 min, taking a reading every minute until 20 minutes, then every 20 seconds. After doing 2 repetitions of pH 8, one of 7, and one of 5, it was determined that the protein had crashed out of the solution and was not reacting with the PNPA.

The ligand used for testing was p-nitrophenyl acetate (PNPA) in pH's of 4, 5, 6, 7, and 8. In each cuvette, 5 µL of protein, 50 µL of ligand, and 1.5 mL buffer (varying pH) were added. Absorbance was measured at 405 nm for 30 min, taking a reading every minute until 20 minutes, then every 20 seconds. After doing 2 repetitions of pH 8, one of 7, and one of 5, it was determined that the protein had crashed out of the solution and was not reacting with the PNPA.

==Discussion==

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== Conclusions ==

The structure of 3DS8 was honed in on after matching the sequence with various other proteins in databases such as BLAST, SPRITE, and DALI. The sequence matches gave information about the active site and possible functions of the protein. The structure was then determined by the sequence in programs such as Chimera, InterPro, and SwissDock. The combination of this research suggested that the 3DS8 protein is a hydrolase. Gel electrophoresis confirmed that the protein was obtained as a dark band was visualized around 30 kDa. Various ligands that are common for hydrolysis were examined and modeled, and the best were used in the lab to gather data. Unfortunately, many of the ligands were on backorder and only PNPA was able to be used in activity assays. No data about the activity was obtained, as all the purified protein crashed out of solution before the assay could be run.

The structure of 3DS8 was honed in on after matching the sequence with various other proteins in databases such as BLAST, SPRITE, and DALI. The sequence matches gave information about the active site and possible functions of the protein. The structure was then determined by the sequence in programs such as Chimera, InterPro, and SwissDock. The combination of this research suggested that the 3DS8 protein is a hydrolase. Gel electrophoresis confirmed that the protein was obtained as a dark band was visualized around 30 kDa. Various ligands that are common for hydrolysis were examined and modeled, and the best were planned to be used in the lab to gather data. Unfortunately, many of the ligands were on backorder and only PNPA was able to be used in enzymatic activity assays. No data about the activity was obtained, as all the purified protein crashed out of solution before the assay could be run.

== References ==

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Listeria innocua using Computational and In-Vitro Techniques. https://www.cell.com/biophysj/pdf/S0006-3495(18)31671-0.pdf

2. http://211.25.251.163/sprite/

2. Mindrebo, J. T.; Nartey, C. M.; Seto, Y.; Burkart, M. D.; Noel, J. P. Unveiling the functional diversity of the alpha/beta hydrolase superfamily in the plant kingdom. Science Direct. 2016, 41, 233-246. DOI: 10.1016/j.sbi.2016.08.005.

3. http://211.25.251.163/sprite/

3. https://www.cgl.ucsf.edu/chimera/download.html

4. https://www.cgl.ucsf.edu/chimera/download.html

4. http://ekhidna2.biocenter.helsinki.fi/dali/

5. http://ekhidna2.biocenter.helsinki.fi/dali/

5. https://blast.ncbi.nlm.nih.gov/Blast.cgi

6. https://blast.ncbi.nlm.nih.gov/Blast.cgi

6. https://www.ebi.ac.uk/interpro/

7. https://www.ebi.ac.uk/interpro/

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@@ Line 15: / Line 15: @@
 There have been previous studies conducted on the 3DS8 protein concluding that it is an alpha-beta hydrolase and originates from the Lin2722 gene in ''Listeria innocua'', a species of Gram-positive bacteria. Studies included database searches and the measurement of enzymatic activity by UV-Vis colorimetric assays using p-nitrophenyl analogs to form p-nitrophenol.[1]
+The αβ-hydrolases found in plants have many functions, including ligand receptors and catalytic activity. They are also adaptable and malleable regarding the fold of the protein secondary structure, which makes them an ideal target for research. [2]
 </StructureSection>
+== Materials ==
+- Buffers: Sodium Phosphate buffer, Cell Lysis Buffer Tris-HCl, 10X SDS-PAGE Buffer, Re-Suspension Buffer, 1X Wash Buffer, 1X Elution Buffer
+- Solutions for SDS-Page: Coomassie Blue Stain, and Destain
+- Pre-cast SDS-Page Gel
 == Experimental and Results ==
-The beginning experiments were conducted on molecular docking sites to compare the structure of the 3DS8 active site with known proteins. Using the SPRITE database [2], the 3DS8 protein was matched with a few proteases through right-handed superpositions with RMSD values ranging from 1.20-1.40, indicating relative similarities.
+The beginning experiments were conducted on molecular docking sites to compare the structure of the 3DS8 active site with known proteins. Using the SPRITE database [3], the 3DS8 protein was matched with a few proteases through right-handed superpositions with RMSD values ranging from 1.20-1.40, indicating relative similarities.
 The left-handed superpositions matched with more trypsins and displayed better RMSD values ranging from 0.93-1.11.
-The 3DS8 protein did not have many very specific active site matches with known proteins on the Chimera software [3], however, it was very similar to trypsin,
+The 3DS8 protein did not have many very specific active site matches with known proteins on the Chimera software [4], however, it was very similar to trypsin,
 alpha-chymotrypsin, and proteinase B. The function of 3DS8 is most likely very similar to those since the active sites have the same amino acids and structures that differ within <4 angstroms.
-The Dali database [4] was used to determine the conserved sequences within the 3DS8 protein. The majority of the hits were lipases with Z-scores up to 31.8, meaning the proteins are homologous
+The Dali database [5] was used to determine the conserved sequences within the 3DS8 protein. The majority of the hits were lipases with Z-scores up to 31.8, meaning the proteins are homologous
 to the 3ds8 structure because they are higher than 20. Some hits had high LALI numbers that indicate matching residues in the structure. The active site residues are G104, S102, H222, D188. Each of
 these is conserved in the 4 selected protein matches, which means that the active site is conserved. The 3ds8 active site is conserved in many other proteins with similar functions, many of which
@@ Line 32: / Line 42: @@
-The BLAST database [5] was utilized to search for similar gene sequences and corresponding residue patterns. Using the FASTA sequence, the 3DS8 sequence is matched with similar proteins in an
+The BLAST database [6] was utilized to search for similar gene sequences and corresponding residue patterns. Using the FASTA sequence, the 3DS8 sequence is matched with similar proteins in an
 alignment that shows similar positions of matching residues. The superfamily of 3DS8 is an αβ-hydrolase. Function and cellular position are unknown, but it is hydrolase-like and exists in bacteria.
 The 3DS8 protein is part of the superfamily of alpha-beta hydrolases, so it most likely has the same function. Many structural and sequential similarities are conserved between 3DS8
@@ Line 43: / Line 53: @@
-InterPro Scan [6] searched for structure and taxonomy relations. The results have information about the domains and families of the protein. At the bottom, there are biological processes,
+InterPro Scan [7] searched for structure and taxonomy relations. The results have information about the domains and families of the protein. At the bottom, there are biological processes,
 molecular functions, and cellular components to learn more about the protein and where it originates from. InterPro confirmed that 3DS8 is most likely a hydrolase. Since 3DS8 is part of
 the hydrolase superfamily, its structure and function are likely to be that of some sort of hydrolase.
@@ Line 59: / Line 69: @@
-Fractions E1 through E6 were chosen to be run on an SDS-PAGE gel because they contained the highest amounts of protein. After running for an hour on 100-150 mV, the gel was stained and destained. The bands appeared around 30 kDa and fractions E2-E6 were pure.
+Fractions E1 through E6 were chosen to be run on an SDS-PAGE gel because they contained the highest amounts of protein. After running for an hour on 100-150 mV, the gel was stained and destained. The bands appeared around 30 kDa and fractions E2-E6 were pure. The controls include running samples from before and after the column and wash.
-The ligand used for testing was p-nitrophenyl aldehyde (PNPA) in pH's of 4, 5, 6, 7, and 8. In each cuvette, 5 µL of protein, 50 µL of ligand, and 1.5 mL buffer (varying pH) were added. Absorbance was measured at 405 nm for 30 min, taking a reading every minute until 20 minutes, then every 20 seconds. After doing 2 repetitions of pH 8, one of 7, and one of 5, it was determined that the protein had crashed out of the solution and was not reacting with the PNPA.
+The ligand used for testing was p-nitrophenyl acetate (PNPA) in pH's of 4, 5, 6, 7, and 8. In each cuvette, 5 µL of protein, 50 µL of ligand, and 1.5 mL buffer (varying pH) were added. Absorbance was measured at 405 nm for 30 min, taking a reading every minute until 20 minutes, then every 20 seconds. After doing 2 repetitions of pH 8, one of 7, and one of 5, it was determined that the protein had crashed out of the solution and was not reacting with the PNPA.
 ==Discussion==
@@ Line 70: / Line 80: @@
 == Conclusions ==
-The structure of 3DS8 was honed in on after matching the sequence with various other proteins in databases such as BLAST, SPRITE, and DALI. The sequence matches gave information about the active site and possible functions of the protein. The structure was then determined by the sequence in programs such as Chimera, InterPro, and SwissDock. The combination of this research suggested that the 3DS8 protein is a hydrolase. Gel electrophoresis confirmed that the protein was obtained as a dark band was visualized around 30 kDa. Various ligands that are common for hydrolysis were examined and modeled, and the best were used in the lab to gather data. Unfortunately, many of the ligands were on backorder and only PNPA was able to be used in activity assays. No data about the activity was obtained, as all the purified protein crashed out of solution before the assay could be run.
+The structure of 3DS8 was honed in on after matching the sequence with various other proteins in databases such as BLAST, SPRITE, and DALI. The sequence matches gave information about the active site and possible functions of the protein. The structure was then determined by the sequence in programs such as Chimera, InterPro, and SwissDock. The combination of this research suggested that the 3DS8 protein is a hydrolase. Gel electrophoresis confirmed that the protein was obtained as a dark band was visualized around 30 kDa. Various ligands that are common for hydrolysis were examined and modeled, and the best were planned to be used in the lab to gather data. Unfortunately, many of the ligands were on backorder and only PNPA was able to be used in enzymatic activity assays. No data about the activity was obtained, as all the purified protein crashed out of solution before the assay could be run.
 == References ==
@@ Line 77: / Line 87: @@
 Listeria innocua using Computational and In-Vitro Techniques. https://www.cell.com/biophysj/pdf/S0006-3495(18)31671-0.pdf
-. http://211.25.251.163/sprite/
+. Mindrebo, J. T.; Nartey, C. M.; Seto, Y.; Burkart, M. D.; Noel, J. P. Unveiling the functional diversity of the alpha/beta hydrolase superfamily in the plant kingdom. Science Direct. 2016, 41, 233-246. DOI: 10.1016/j.sbi.2016.08.005.
+. http://211.25.251.163/sprite/
 . https://www.cgl.ucsf.edu/chimera/download.html
 . http://ekhidna2.biocenter.helsinki.fi/dali/
 . https://blast.ncbi.nlm.nih.gov/Blast.cgi
 . https://www.ebi.ac.uk/interpro/