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2025-04-27T01:15:49Z

Emma Caszatt:

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2025-04-27T01:15:15Z

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2025-04-27T01:14:04Z

Emma Caszatt:

Characterization and Preliminary Functionality of 3B7F

3B7F is a currently unknown protein in terms of its function. Based on current structural analysis, it consists of one unique chain with a mass of 43.34 kDa and an atom count of 3,216. The mass was found by multiply the 394 amino acids of the protein by the mass of each amino acid (~0.110 kDa). Based on previous studies, 3B7F is assumed to be a glycosyl hydrolase, however, the function is still not entirely known.[1] Through the following procedures and data collection, the goal of this research was to analyze the sequence and structure of 3B7F in order to better understand its enzymatic function.

'''Research Question:''' What is the function of the 3B7F protein, and how can this be determined through both computational and wet lab techniques?

'''Relevance:''' The goal of this research is to determine the function of the 3BF7 protein in order to evaluate whether it can degrade xyloglucan or other types of carbohydrates and glycoconjugates in plants. Knowing this will allow future researchers to be able to better understand xyloglucan/carbohydrate glycoconjugate degradation in plants, and allow for known pathways to be expanded upon. Through this experimentation, we can also learn more about the ways in which both computational bioinformatics and wet lab techniques can aid in determining the function of a protein with a known structure.

'''Hypothesis:''' 3B7F is a xyloglucanase, a type of glycosyl hydrolase that acts to degrade xyloglucan in plant cell walls. It presents optimal activity in fairly acidic conditions and demonstrates potentially satisfactory binding with PNP phosphate and lysine p nitroanilide.

<StructureSection load='3B7F' size='400' side='right' caption='Structure of 3B7F' scene=''>

== Methods ==

'''SPRITE:'''

The PBD ID for the protein of interest (3B7F) was entered and 2-residue hits excluded. Once the search was complete, "List of Hits" results were obtained. "Full details" result alignments also analyzed. Hits by each side of protein viewed by clicking "Arranged by sites" function. Alignments with an RMSD below 2.0 Angstroms were reviewed.

'''Chimera:
'''
PBD ID of protein of interest entered and “fetch” was selected in order to show the structure of the protein. Based on the results from SPRITE, the protein of known function was loaded into Chimera using its PBD ID. Everything but the subunit of interest was hidden, and then the active site motif was aligned. The RMSD value was shown and used to determine the quality of alignment (anything below 2.0 Angstroms was considered high quality). To better visualize the alignment, "match" was deleted and replaced with "sel".

'''Dali:
'''
PDB search tab was selected, and the four-letter PDB of the assigned protein (3B7F) was entered. The structure with the chain identifier was submitted. The job was viewed after completion.

'''BLAST:
'''
The PDB ID was searched for in the RCSB webpage. "FASTA sequence” was selected and the protein sequence was copied into the NCBI BLAST search page. Proteins similar to the query protein were identified based on sequence.

'''InterPro:
'''
An InterPro search was performed for the sequence of 3B7F. Protein superfamily identification and domains were reviewed. Related proteins in the domain organization were identified. The structures were also analyzed.

'''SwissDock:
'''
The "Docking with AutoDock Vina" tab was selected on SwissDock. A ligand by submitted by using SMILES string (found through the PubChem database). "Prepare ligand" was clicked, and a target (can use PDB ID) was submitted. The search space was defined, and x, y, and z coordinates for the center of the space being searched were chosen. The parameters were checked and docking was started. Results were analyzed.

'''Buffers and Solutions:
'''
General steps for Buffers and solutions included adding 80% of total DiH2O to a container, weighing and adding chemicals, adjusting pH as needed, and adding DiH2O to the total volume.

'''Hand Casting Polyacrylamide Gels:
'''
Made a 4% stacking gel at a pH of 6.8, and a 10% resolving gel at a pH of 8.8.

Resolving and stacking gel solutions were prepared without APS or TEMED. A comb was placed into the assembled gel sandwich with a marker. A mark was placed on the glass plate 1 cm below the teeth of the comb, and the comb was removed. The APS and TEMED were added to the resolving gel, and the solution was poured to the mark. The gel was allowed to polymerize for 45-60 minutes. APS and TEMED were added to the stacking solution and poured above the resolving gel. The comb was placed in the cassette and tilted so that the teeth are at a 10º angle. This prevented air from becoming trapped under the comb. The gel was allowed to polymerize for 30-45 minutes. The gels were wrapped in a wet paper towel in the 4ºC fridge for storage.

'''Expression of Proteins from Lactose-Inducible Vectors:
'''
The LB Broth was made by adding 10g of tryptone, 10g of NaCl, and 5g of yeast extract together. This was added to 1000 mL of Millipore water. Five mL of this mixture was poured into an overnight culture tube. The other broth was autoclaved for 1.5 hours.

'''Protein Purification:
'''
500 mL of the protein was grown rather than 1L in an attempt to speed up induction. The overnights were grown at night on 03/16/2025 for 11 hours with 50 µg/mL of kanamycin. It was inoculated at 8 am on 03/17/2025. OD600 nm was taken in the morning until 0.4-0.8 OD (an OD of 0.4 was reached around 10 am). It was then induced with 1 nM IPTG and left to grow for three hours before centrifuging. In order to purify, the samples were centrifuged at 5000 x g for 20 minutes. Ten mL of lysis buffer was added, and the pellets were resuspended with a pipette (50 µL was added to a separate centrifuge tube). The cells were sonicated 5x for 30 seconds on ice in between each sonication (50 µL of a sample was added to a new centrifuge tube). The samples were centrifuged for 20 minutes at 15,000 x g (50 µL of a sample was added to a new centrifuge tube). The protein column was set up with 500 µL Ni-NTA beads and a lysis buffer was ran through it to equilibrate it (5x column volumes). The resin was pre-washed with a binding buffer. The cell extract was applied to the resin and allowed to enter. All of the supernatant was added (50 µL of a sample was added to a new centrifuge tube). The column was washed with 5 column volumes of buffer (50 µL of a sample was added to a new centrifuge tube). The column was eluted with 8 column volumes of buffer, the fractions were collected in 1 mL volumes and stored in 5 tubes. To store the column, 5 column volumes of water, and 1-2 mL of 20% EtOH were added, and the column was capped off and stored.

'''Protein Concentration:
'''
Seven BSA standards were prepared using the buffer that the protein was stored in (elution buffer). One mL of Bradford Reagent was added to each cuvette. Twenty µL of water was added to the zero cuvette, while 20 µL of each BSA standard or elution was added to the rest of cuvettes. The cuvettes were covered with parafilm and mixed several times by inversion. They were maintained at room temperature for 5-45 minutes. Absorbance was recorded at 595 nm with a Vernier spectrophotometer. A standard curve was constructed and used to find the concentration of the unknown protein in each elution.

'''SDS-PAGE:
'''
For sample prep, the protein samples were treated with SDS sample buffer and boiled before application. The final concentration of SDS sample buffer loaded onto the gel was 1x.

The set up was loaded with 1x running buffer. The ladder and samples were then loaded into the lanes (20 µL of samples loaded). The protective cover and cables were attached and connected to the power supply. The gel was run at a constant voltage of 120V. The gel was run until the sample line was about 1 cm from the bottom of the gel.

In order to stain the gel, it was removed from the gel sandwich and gently added to a shallow plastic container. InstantBlue stain was added to cover the gel, and it was agitated overnight. The stain was removed and the destaining solution added. The gel incubated for 30 minutes with a paper towel. It was then rinsed with water and captured on the gel imager.

'''Protein Activity Assay:
'''
Three mL of buffer and 3 mg of substrate (PNPP or PNPA) were added to a cuvette. It was then placed in the spectrophotometer, and the instrument was zeroed at 405 nm. The desired amount of the protein of interest (25-75 uL) was added, and the solution was mixed quickly. The absorbance at 405 nm was read over time. Measurements were taken every minute until a change was observed, and then measurements were taken every 30 seconds.

== Structural Alignment Through SPRITE, Chimera, Dali, and BLAST ==

Based on the findings through SPRITE and Chimera, 1XNY_C00 had the lowest RMSD value at 1.875 angstroms. Therefore, it is hypothesized that 3B7F was a carboxylase.

[[Image:SPRITE.jpg]] [[Image:Chimera.jpg]]
A 183 GLY matches A 207 GLY
B 419 GLY matches A 154 GLY
B 420 ALA matches A 153 ALA

'''Figure 1.''' SPRITE and Chimera binding of 3B7F (yellow) with 1XNY_C00 (green).

Dali provided mainly xyloglucanase matches and no matches were carboxylases. Therefore, the initial hypothesis that 3B7F was a carboxylase was no longer supported. Based on the Dali results, it is now hypothesized that 3B7F is a xyloglucanase. [2]

'''Table 1.''' Top Matches on Dali (Z: greater than 4.0 indicates structural significance; RMSD: root mean square deviation (distance in Angstroms between superimposed molecules); LALI: length of alignment; NRES: number of residues in hit structure; %ID: similarity between hit sequence and query sequence).
[[Image:TableDali.jpg]]

[[Image:BLAST.jpg]]
Figure 2.''' Superimposition of 6P2N:A (orange) and 3B7F (green).
'''

Xyloglucanases break down xyloglucan, a hemicellulose in plant cell walls. The substrate is xyloglucan and water for the (endo-)beta-1,4-xyloglucanases, and a common cofactor is calcium. [3]

The BLAST search shows glycosyl hydrolases having similar sequences to 3B7F. The xyloglucanases/glycosyl hydrolases from the BLAST search come from Cupriavidus, a type of gram-negative bacteria. The E-values were 0.0 for most of the results, signifying high matches. The scores were high as well, with percent identification in the 90 percentage range. The query coverage was also 100% for most of the results. Based on the results, the protein family of 3B7F is likely sialidase, which breaks down sialic acid into carbohydrates. Sialidases are a family of proteins that include glycosyl hydrolases and xyloglucanases, which further supports the hypothesis that 3B7F is a xyloglucanase. [4]

== Using InterPro to Predict Protein Function ==

The family with the closest similarities to 3B7F is xyloglucanase, which matches the BLAST and Dali findings. These enzymes act on xyloglucan, a plant wall polysaccharide, by breaking the glucosidic bonds of unbranched glucose residues. Most of the proteins in this family bind to cell wall structures or participate in photosystem II, which correlates with the expected activity of 3B7F, since it would be acting on plant cell walls. This family consists mainly of bacteria, as well as eukaryotes and archaea. This also matches the BLAST data because it suggested that the sequence was commonly found in bacteria. The proteomes are a lot of bacteria and marine life. The most closely matched structures are different xyloglucanases and oligoxyloglucan reducing end-specific cellobiohydralases, which is the superfamily that the protein belongs to. The main pathways are also structural degradation pathways, which also aligns with the results we have gained so far. [5]

== Molecular Docking with SwissDock ==

Based on the hydrolase ligands that we analyzed, PNP phosphate seems to be the best ligand for 3B7F, with the highest binding affinity of -8.189 kcal/mol, and the lowest binding affinity of -6.146 kcal/mol. This is a very strong binding affinity, so this substrate was used for protein assays. Lysine p nitroanilide also has its highest binding affinity at -7.286 kcal/mol and the lowest binding affinity of -6.551 kcal/mol, indicating that it is another suitable substrate for 3B7F. [6]

[[Image:SwissDock1.jpg]]
'''Figure 3.''' 3B7F Binding with PNPP at -8.189 kcal/mol.

== Protein Concentration ==

Based on the Bradford Assay, elution 4 had the most protein (4.118 mg/mL), while elution 3 had the second most amount of protein (3.261 mg/mL). The rest of the elutions had protein concentrations around 2 mg/mL. The R² value of the standard curve was 0.9857, indicating fairly accurate results.

[[Image:StandardCurve.jpg]]
'''Figure 4.''' Standard Curve at 595 nm for 3B7F Elutions.

'''Table 2.''' Final Protein Concentration of Each Elution
[[Image:FinalConc.jpg]]

== Protein Analysis ==

Possibly due to errors during purification, no bands were seen on the SDS-PAGE gel for 3B7F.

Several conditions were analyzed through enzyme activity assays. Initially, 50 uL of 3B7F elution 4 with 1 mg/mL PNPP demonstrated some linearity between 0-600 seconds. However, this was not able to be replicated. Both 25 uL and 75 uL of 3B7F elution 4 were also used with PNPP, and no linearity was found. Due to the enzyme’s optimal conditions, it was also cooled down in -20°C for five minutes, however, no linearity was seen. The assay was also perfomed with elution 1 to identify if any protein was present after the wash. Again, no linearity was shown. PNPA was also used as a substrate with elution 4, and pHs of 1.5 and 5.0 were also used during separate trials in order to identify the optimal conditions of the enzyme. [7] However, these assays still showed no linearity. It was determined that there was an error during purification, leading to unidentifiable concentrations of 3B7F for enzymatic study.

[[Image:Assay.jpg]]
'''Figure 5.''' 50 uL of 3B7F Elution 4 with 1 mg/mL PNPP.

[[Image:Assay2.jpg]]
Slope = 7 x 10⁻⁵ Abs/sec
C = 7 x 10⁻⁵/18000*1
= 3.89 x 10⁻⁹ M/sec
'''Figure 6.''' Zoom in with linear trendline of Figure 5 and enzyme activity calculations.

== Conclusion ==
Based on the results, no enzymatic function of 3B7F can be appropriately determined. However, based on the computational results, it is likely that 3B7F has xyloglucanase activity. In the future, the protein should be grown again in order to retest the protein’s enzymatic activity after a proper purification. The results that were successfully gathered did show accuracy and precision. However, likely due to incorrect solution making, the protein was not able to be properly purified, and therefore, was not present in the elutions. Interestingly enough, the Bradford Assay did show protein concentrations, but they likely were not for the desired protein (3B7F), which may be the reason for no protein present at the expected mass of approximately 43 kDa. If 3B7F is confirmed as a xyloglucanase, it can possibly be used for drug targeting in the colon through xyloglucan coatings. [8]

</StructureSection>

== References ==

[1] RCSB PDB - 3B7F: Crystal structure of a putative glycosyl hydrolase with bnr repeats (reut_b4987) from ralstonia eutropha jmp134 at 2.20 A resolution. Rcsb.org. https://www.rcsb.org/structure/3b7f (accessed 2025-03-09).

[2] Dali server. ekhidna2.biocenter.helsinki.fi. http://ekhidna2.biocenter.helsinki.fi/dali/.

[3] Glycoside hydrolases - CAZypedia. www.cazypedia.org. https://www.cazypedia.org/index.php/Glycoside_hydrolases.

[4] NCBI. BLAST: Basic Local Alignment Search Tool. Nih.gov. https://blast.ncbi.nlm.nih.gov/Blast.cgi.

[5] InterPro EMBL-EBI. InterPro protein sequence analysis & classification < InterPro < EMBL-EBI. Ebi.ac.uk. https://www.ebi.ac.uk/interpro/.

[6] SwissDock. www.swissdock.ch. https://www.swissdock.ch/.

[7]Shi, H.; Guo, J.; Yan, X.; Cui, G.; Tan, Z.; Zhu, X.; Zhou, J.; He, S.; Wang, T.; Li, X. Characterization of a Xyloglucananse in Biodegradation of Woody Plant Xyloglucan from Caldicellulosiruptor Kronotskyensis. BioResources 2021, 17 (1), 673–681. https://doi.org/10.15376/biores.17.1.673-681.

[8] Doggwiler, V.; Lanz, M.; Paredes, V.; Lipps, G.; Imanidis, G. Tablet Formulation with Dual Control Concept for Efficient Colonic Drug Delivery. International Journal of Pharmaceutics 2023, 631, 122499. https://doi.org/10.1016/j.ijpharm.2022.122499.

<references/>

File:FinalConc.jpeg

2025-04-27T01:13:16Z

Emma Caszatt:

File:TableDali.jpeg

2025-04-27T01:12:37Z

Emma Caszatt:

2025-04-27T00:25:10Z

Emma Caszatt:

Characterization and Preliminary Functionality of 3B7F

3B7F is a currently unknown protein in terms of its function. Based on current structural analysis, it consists of one unique chain with a mass of 45.04 kDa and an atom count of 3,216. Based on previous studies, 3B7F is assumed to be a glycosyl hydrolase, however, the function is still not entirely known.[1] Through the following procedures and data collection, the goal of this research was to analyze the sequence and structure of 3B7F in order to better understand its enzymatic function.

'''Research Question:''' What is the function of the 3B7F protein, and how can this be determined through both computational and wet lab techniques?

'''Relevance:''' The goal of this research is to determine the function of the 3BF7 protein in order to evaluate whether it can degrade xyloglucan or other types of carbohydrates and glycoconjugates in plants. Knowing this will allow future researchers to be able to better understand xyloglucan/carbohydrate glycoconjugate degradation in plants, and allow for known pathways to be expanded upon. Through this experimentation, we can also learn more about the ways in which both computational bioinformatics and wet lab techniques can aid in determining the function of a protein with a known structure.

'''Hypothesis:''' 3B7F is a xyloglucanase, a type of glycosyl hydrolase that acts to degrade xyloglucan in plant cell walls. It presents optimal activity in fairly acidic conditions and demonstrates potentially satisfactory binding with PNP phosphate and lysine p nitroanilide.

<StructureSection load='3B7F' size='400' side='right' caption='Structure of 3B7F' scene=''>

== Methods ==

'''SPRITE:
'''
The PBD ID for the protein of interest (3B7F) was entered and 2-residue hits excluded. Once the search was complete, "List of Hits" results were obtained. "Full details" result alignments also analyzed. Hits by each side of protein viewed by clicking "Arranged by sites" function. Alignments with an RMSD below 2.0 Angstroms were reviewed.

'''Chimera:
'''
PBD ID of protein of interest entered and “fetch” was selected in order to show the structure of the protein. Based on the results from SPRITE, the protein of known function was loaded into Chimera using its PBD ID. Everything but the subunit of interest was hidden, and then the active site motif was aligned. The RMSD value was shown and used to determine the quality of alignment (anything below 2.0 Angstroms was considered high quality). To better visualize the alignment, "match" was deleted and replaced with "sel".

'''Dali:
'''
PDB search tab was selected, and the four-letter PDB of the assigned protein (3B7F) was entered. The structure with the chain identifier was submitted. The job was viewed after completion.

'''BLAST:
'''
The PDB ID was searched for in the RCSB webpage. "FASTA sequence” was selected and the protein sequence was copied into the NCBI BLAST search page. Proteins similar to the query protein were identified based on sequence.

'''InterPro:
'''
An InterPro search was performed for the sequence of 3B7F. Protein superfamily identification and domains were reviewed. Related proteins in the domain organization were identified. The structures were also analyzed.

'''SwissDock:
'''
The "Docking with AutoDock Vina" tab was selected on SwissDock. A ligand by submitted by using SMILES string (found through the PubChem database). "Prepare ligand" was clicked, and a target (can use PDB ID) was submitted. The search space was defined, and x, y, and z coordinates for the center of the space being searched were chosen. The parameters were checked and docking was started. Results were analyzed.

'''Buffers and Solutions:
'''
General steps for Buffers and solutions included adding 80% of total DiH2O to a container, weighing and adding chemicals, adjusting pH as needed, and adding DiH2O to the total volume.

'''Hand Casting Polyacrylamide Gels:
'''
Made a 4% stacking gel at a pH of 6.8, and a 10% resolving gel at a pH of 8.8.

Resolving and stacking gel solutions were prepared without APS or TEMED. A comb was placed into the assembled gel sandwich with a marker. A mark was placed on the glass plate 1 cm below the teeth of the comb, and the comb was removed. The APS and TEMED were added to the resolving gel, and the solution was poured to the mark. The gel was allowed to polymerize for 45-60 minutes. APS and TEMED were added to the stacking solution and poured above the resolving gel. The comb was placed in the cassette and tilted so that the teeth are at a 10º angle. This prevented air from becoming trapped under the comb. The gel was allowed to polymerize for 30-45 minutes. The gels were wrapped in a wet paper towel in the 4ºC fridge for storage.

'''Expression of Proteins from Lactose-Inducible Vectors:
'''
The LB Broth was made by adding 10g of tryptone, 10g of NaCl, and 5g of yeast extract together. This was added to 1000 mL of Millipore water. Five mL of this mixture was poured into an overnight culture tube. The other broth was autoclaved for 1.5 hours.

'''Protein Purification:
'''
500 mL of the protein was grown rather than 1L in an attempt to speed up induction. The overnights were grown at night on 03/16/2025 for 11 hours with 50 µg/mL of kanamycin. It was inoculated at 8 am on 03/17/2025. OD600 nm was taken in the morning until 0.4-0.8 OD (an OD of 0.4 was reached around 10 am). It was then induced with 1 nM IPTG and left to grow for three hours before centrifuging. In order to purify, the samples were centrifuged at 5000 x g for 20 minutes. Ten mL of lysis buffer was added, and the pellets were resuspended with a pipette (50 µL was added to a separate centrifuge tube). The cells were sonicated 5x for 30 seconds on ice in between each sonication (50 µL of a sample was added to a new centrifuge tube). The samples were centrifuged for 20 minutes at 15,000 x g (50 µL of a sample was added to a new centrifuge tube). The protein column was set up with 500 µL Ni-NTA beads and a lysis buffer was ran through it to equilibrate it (5x column volumes). The resin was pre-washed with a binding buffer. The cell extract was applied to the resin and allowed to enter. All of the supernatant was added (50 µL of a sample was added to a new centrifuge tube). The column was washed with 5 column volumes of buffer (50 µL of a sample was added to a new centrifuge tube). The column was eluted with 8 column volumes of buffer, the fractions were collected in 1 mL volumes and stored in 5 tubes. To store the column, 5 column volumes of water, and 1-2 mL of 20% EtOH were added, and the column was capped off and stored.

'''Protein Concentration:
'''
Seven BSA standards were prepared using the buffer that the protein was stored in (elution buffer). One mL of Bradford Reagent was added to each cuvette. Twenty µL of water was added to the zero cuvette, while 20 µL of each BSA standard or elution was added to the rest of cuvettes. The cuvettes were covered with parafilm and mixed several times by inversion. They were maintained at room temperature for 5-45 minutes. Absorbance was recorded at 595 nm with a Vernier spectrophotometer. A standard curve was constructed and used to find the concentration of the unknown protein in each elution.

'''SDS-PAGE:
'''
For sample prep, the protein samples were treated with SDS sample buffer and boiled before application. The final concentration of SDS sample buffer loaded onto the gel was 1x.

The set up was loaded with 1x running buffer. The ladder and samples were then loaded into the lanes (20 µL of samples loaded). The protective cover and cables were attached and connected to the power supply. The gel was run at a constant voltage of 120V. The gel was run until the sample line was about 1 cm from the bottom of the gel.

In order to stain the gel, it was removed from the gel sandwich and gently added to a shallow plastic container. InstantBlue stain was added to cover the gel, and it was agitated overnight. The stain was removed and the destaining solution added. The gel incubated for 30 minutes with a paper towel. It was then rinsed with water and captured on the gel imager.

'''Protein Activity Assay:
'''
Three mL of buffer and 3 mg of substrate (PNPP or PNPA) were added to a cuvette. It was then placed in the spectrophotometer, and the instrument was zeroed at 405 nm. The desired amount of the protein of interest (25-75 uL) was added, and the solution was mixed quickly. The absorbance at 405 nm was read over time. Measurements were taken every minute until a change was observed, and then measurements were taken every 30 seconds.

== Structural Alignment Through SPRITE, Chimera, Dali, and BLAST ==

Based on the findings through SPRITE and Chimera, 1XNY_C00 had the lowest RMSD value at 1.875 angstroms. Therefore, it is hypothesized that 3B7F was a carboxylase.

[[Image:SPRITE.jpg]]

Dali gave mainly xyloglucanase matches and no matches were carboxylases, the hypothesis that 3B7F was a carboxylase was proven to be wrong. It is not hypothesized that 3B7F is a xyloglucanase. This was due to the fact that 1XNY did not show up as a match in Dali.

Xyloglucanases break down xyloglucan, a hemicellulose in plant cell walls. The substrate is xyloglucan and water for the (endo-)beta-1,4-xyloglucanases, and a common cofactor is calcium.

The BLAST search shows glycosyl hydrolases having similar sequences to 3B7F.

== Using InterPro to Predict Protein Function ==

Research shows that 4Q7Q is a member of the SGNH Hydrolase protein super family. BLAST and InterPro both suggested 4Q7Q’s inclusion in this family, and the known conserved residues seen from SPRITE analysis—Serine, Glycine, Asparagine, and Histidine—line up with those observed throughout this family.D,E Notably, this superfamily is also referred to as the GDSL Hydrolase superfamily.D,E

4Q7Q’s inclusion in this family also supports its SPRITE-derived hypothetical functionality. Rhamnogalacturonan Acetylesterase—the enzyme with one of the best SPRITE-based alignment relative to 4Q7Q—is a member of this family.F Proteins in this family are also known for containing a “unique hydrogen bond network that [stabilizes]” the active site.F

Regarding what protein family 4Q7Q belongs to, DALI results suggest it is a part of a sub-family of the greater GDSL/SGNH superfamily. A PDB90% DALI search labels 4Q7Q as a part of the “Lipolytic Protein G-D-S-L Family,” which refers to enzymes that hydrolyze lipid substrates.I

== Molecular Docking with SwissDock ==

The primary sequence of 4Q7Q shows several conserved sequences between it and esterase-like proteins. A sequence of GDSI—similar to the GDSL sequence seen from its family and superfamily—can be seen between 4Q7Q and enzymes like Isoamyl Acetate-Hydrolyzing Esterase. Other noteworthy conserved sequences between esterases and 4Q7Q include GxND and DGxH.

These enzymes also share similar secondary structures. Segments of alpha-helixes and beta-sheet strands appear and remain nearly entirely conserved throughout esterase analysis. A few conserved coils appear, but these sections do not appear as often as the other two secondary structures.

Similar conserved sequences could be found between 4Q7Q and lipases. The GDSI, GxND, and DGxH sequences can be seen from lipases like 7BXD.? The same secondary structure segments can also be located in the lipases analyzed.

== Protein Purification ==

Right-hand SPRITE analysis revealed 4Q7Q exhibited residues like those seen from enzymes operating with acetyl-like substrates. Specifically, residues Ser. 30, Gly. 69, Asn. 97, Asp. 251, and His. 254 on the A and B chains of 4Q7Q line up with similarly positioned residues on esterases like Platelet-Activating Factor Acetylhyd (PAFA), which exhibited an RMSD of 0.25 angstroms when compared to 4Q7Q.

Other proteins with similar motifs of note are Thioesterase I and Rhamnogalacturonan Acetylesterase, with RMSD values of 0.46 and 0.61, respectively. These alignments focus on the same active site as PAFA did, suggesting the acetyl-like substrates 4Q7Q focuses on are similar to esters.

PFAM graphics from DALI revealed significant structural equivalence between 4Q7Q, a lipase-like protein, Rhamnogalacturonan Acetylesterase, and Sialate O-acetylesterase.

== Protein Concentration ==

SwissDock analysis showed a preference for larger molecules, specifically fatty acids. Lactide, Ethyl Butyrate, and Triethylene Glycol exhibited noticeably weak binding affinities to the theorized active site of 4Q7Q. These ligands may be ill-suited to act as substrates for 4Q7Q as they are remarkably polar, and lipids—one of the potential categories of substrates for 4Q7Q—are mostly non-polar.

Despite this, these ligands show noticeable hydrophobic interactions with the active site. This implies 4Q7Q uses hydrophobic regions to help guide substrates into the right orientation for enzymatic processes. This also further supports the possibility that 4Q7Q primarily operates with hydrophobic lipid-based substrates. This also explains why Methyl Acetate exhibited a relatively weaker affinity for 4Q7Q, as its smaller structure prevented hydrophobic interactions.

== Protein Analysis ==

Highlight the data that helped you come to your conclusion here including any relevant figures. Make sure include potential substrates and binding sites.

</StructureSection>

== References ==

A) 1WAB. Protein Database, 1997. https://www.rcsb.org/structure/1WAB
B) Ho, Y. S.; Sewnson, L.; Derewenda, U.; Serre, L.; Wei, Y.; Dauter, Z.; Hattori, M.; Adachi, T.; Aoki, J.; Arai, H.; Inoue, K.; Derewenda, Z. S. Brain acetylhydrolase that inactivates platelet-activating factor is a G-protein-like trimer. Nature, 1997, 385, 89-93. https://www.nature.com/articles/385089a0 https://www.nature.com/articles/385089a0
C) Miesfeld, R. L.; McEvoy, M. M. Biochemistry, 2nd ed.; W. W. Norton & Company, 2021.
D) SGNH hydrolase superfamily. InterPro, 2017. https://www.ebi.ac.uk/interpro/entry/InterPro/IPR036514/
E) Molgaard, A.; Kauppinen, S.; Larsen, S. Rhamnogalacturonan acetylesterase elucidates the structure and function of a new family of hydrolases. Struct., 2000, 8(4), 373-383. https://www.sciencedirect.com/science/article/pii/S0969212600001180?via%3Dihub
F) 4Q7Q. Protein Database, 2014. https://www.rcsb.org/structure/4Q7Q
G) Rio, T. G. D.; et al. Complete genome sequence of Chitinophaga pinensis type strain (UQM 2034). Stand. Genomic. Sci., 2010, 2(1), 87-95. https://pmc.ncbi.nlm.nih.gov/articles/PMC3035255/
H) Akoh, C. C.; Lee, G.; Liaw, Y.; Huang, T.; Shaw, J. GDSL family of serine esterases/lipases. Prog. Lipid Res., 2004, 43(6), 534-552. https://pubmed.ncbi.nlm.nih.gov/15522763/
I) 7BXD. Protein Database, 2021. https://www.rcsb.org/structure/7BXD
J) Madej,T.; Lanczycki, C. J.; Zhang, D.; Thiessen, P. A.; Geer, R. C.; Marchler-Bauer, A.; Bryant, S. H. MMDB and VAST+: tracking structural similarities between macromolecular complexes. Nucleic Acids Res., 2014, 42(Database), D297-303. https://doi.org/10.1093/nar/gkt1208

<references/>

2025-04-23T17:36:57Z

Emma Caszatt:

==Proposed Structure of 3B7F Protein==

<StructureSection load='3B7F' size='300' side='right' caption='3DS8 Structure' scene=''>
<scene name='10/1043632/3ds8_secondary_structure/1'>3DS8 Secondary Structure</scene>

The discovery and characterization of the structure and function of the protein 3DS8.
This project aimed to characterize the function of an unknown protein 3DS8 with a known structure by comparing it to known proteins and subjecting it to different tests in the laboratory.
Different techniques to achieve this goal include protein expression, purification and analysis, kinetics, and computational methods.

== Function ==
The proposed function of the unknown protein 3DS8 is hydrolase activity. It has many structural similarities to known proteins with hydrolase and protease activity.

The function was not confirmed during wet lab experiments due to protein and reagent complications.

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 [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 [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 [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
are lipases. Since the 3DS8 has so many structural similarities to lipases, it most likely has the same functionality as the known proteins.

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
and matched proteins, indicating that 3DS8 could very well be a hydrolase.

FASTA sequence of 3DS8:
KDQIPIILIHGSGGNASSLDKMADQLMNEYRSSNEALTMTVNSEGKIKFEGKLTKDAKRPIIKFGFEQNQATPDDWSKWLKIAMEDLKSRYGFTQMDGVGHSNGGLALTYYAEDYAGDKTVPTLRKLVAIGSPFND
LDPNDNGMDLSFKKLPNSTPQMDYFIKNQTEVSPDLEVLAIAGELSEDNPTDGIVPTISSLATRLFMPGSAKAYIEDIQVGEDAVHQTLHETPKSIEKTYWFLEKFKTDETVIQLDYK

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.

The last molecular modeling strategy was using SwissDock to investigate different ligands for 3DS8. Using the previously mentioned 4 residues that make up the active site, there were a few
ligands that demonstrated promising binding to 3DS8. The best choices would be PNP alpha-D-glucopyranoside or PNP N-acetyl-Beta-D-glucosaminide because they are close to the residues
of the active site.

The molecular weight of 3DS8 is proposed to be about 28 kDa, based on the length of the amino acid sequence.

In the laboratory, the gene was transformed into ''E. coli'' bacteria, expressed and isolated by cell lysis and nickel column. The cell lysis buffer used was a sodium phosphate buffer. From the column, 10 elution fractions were collected. A Bradford assay with standards of 0.125, 0.25, 0.5, 0.75, and 1 mg/mL of Bovine Serum Albumin solution. The standards were measured on a UV-Vis spectrophotometer at 595 nm. Initially, a few of the elution fractions were out of the standard range of absorbance, so the samples were remade by diluting further. After the second round of absorbances was obtained, only elution 4 was still out of range. Then Beer's law (A=εbc) was used to determine the concentrations (mg/mL) of each elution fraction: E1=0.4026, E2=5.968, E3=5.434, E4=-0.1407, E5=0.5610, E6=0.4229, E7=0.03175, E8=0.1461, E9=0.2693, E10=0.2244.

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 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==

The 3DS8 protein had already been sequenced, so comparisons between sequence and structure were carried out in a variety of programs to discern the possible function. After finding homologous and conserved regions from the databases, it was determined that the protein was an αβ-hydrolase. Once the function was proposed, common ligands were found to be modeled in active site docking software. During the molecular docking studies, PNPA was discovered to be a good ligand with multiple bonds to the active site of 3DS8. The protein vector was purchased and the 3DS8 protein was purified from the bacteria. The amount of protein in each column was quantified using a Bradford Assay to determine the elutions with the highest concentration of protein. Some elutions had a negative amount, due to either being out of range of the plot or instrumental variation. Some of the samples were remade in order to correctly quantify them using the Bradford Assay. To verify the correct protein was purified from the column, gel electrophoresis was run. The gel confirmed a protein around 30 kDa, which was proposed earlier. To assess the activity of the protein, the protein was going to be assessed with the various ligands found in the molecular docking studies using UV-Vis. Many of the other possible substrates, such as PNP Butyrate, were on backorder but PNPA was delivered. The activity of the protein was all baseline, even after having complete saturation of the ligand in protein solution at various pH. After much confusion, it was concluded that the protein was no longer viable. Unfortunately, since the project was done over a couple of weeks, the concentration of protein in the elutions was too high and crashed out. The raw data about the 3DS8 activity was inconclusive.

== 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 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 ==
<references/>
1. Sharkawy, M.; Carter, A.A.; Craig, P. Function Identification of the Protein Product of Gene Lin2722 from
Listeria innocua using Computational and In-Vitro Techniques. https://www.cell.com/biophysj/pdf/S0006-3495(18)31671-0.pdf

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/

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

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

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

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