Sandbox 323
From Proteopedia
Contents |
Proposed Structure of 3DS8 Protein
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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
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/
