Difference between revisions of "Part:BBa K3843001"

Latest revision as of 03:37, 21 October 2021

Affinity-improved PEA-binding salmon trypsin (1UTM+)

Trypsin is a ubiquitous protease in vertebrates due to its central role in digestion. Trypsin cleaves peptide bonds occurring after positively charged amino acids, such as lysine and arginine. 1UTM (pictured below) is a trypsin from Salmo salar (Atlantic salmon). It has a variety of known inhibitors, typically biogenic amines (Leiros et al., 2009). These noncompetitive inhibitors bind to allosteric sites, thereby inactivating trypsin's proteolytic function. Of particular interest to Waterloo iGEM 2021 was the ability of phenylethylamine (PEA, pictured below in green), a dopamine precursor, to act as an inhibitor of 1UTM, with a dissociation constant Kd = 0.000972 (Leiros et al., 2009; RCSB PDB, n.d.). This would allow for 1UTM to be used as a PEA-binding protein. Detection and quantification of phenylethylamine, using 1UTM fused to horseradish peroxidase in a microfluidic flow assay, would give insight into an individual's probability of having ADHD.

Waterloo iGEM 2021 sought to improve the binding affinity of 1UTM to PEA in order to increase the reliability of PEA detection in their diagnostic assay. To do so, they employed a computational approach to rational protein design. First, 1UTM was computationally mutated at every residue to every amino acid. Then, the stability of the mutated protein was assessed through overall energy scoring of the lone protein using Rosetta. Next, the protein's binding affinity to phenylethylamine was assessed using ligand docking in AutoDock Vina as well as molecular dynamics simulations in GROMACS, which returned an energy score for the phenylethylamine+1UTM complex; lower energy scores indicated a higher binding affinity. The mutant with the highest binding affinity, where the mutation was in the active site of the protein, was chosen as the improved version of 1UTM. The mutation and evaluation process was repeated until a triple mutant of 1UTM was obtained, containing the mutations Y204F, C195D, and S190T. This affinity-improved version of 1UTM displayed a decrease in the energy scores of the 1UTM-PEA complex by 2.6 kcal/mol (on AutoDock Vina) and 20.8948 kJ/mol (on GROMACS); both correspond to an increase in binding affinity.

The following image depicts the affinity-improved 1UTM, with 1UTM in orange and PEA in green.

References

Leiros, H. K. S., Brandsdal, B. O., Andersen, O. A., Os, V., Leiros, I., Helland, R., Otlewski, J., Willassen, N. P., & Smalås, A. O. (2009, January 1). Trypsin specificity as elucidated by lie calculations, x‐ray structures, and association constant measurements. Protein Science, 13(4), 1056-1070. https://onlinelibrary.wiley.com/doi/full/10.1110/ps.03498604.

Pettersen, E. F.; Goddard, T. D.; Huang, C. C.; Couch, G. S.; Greenblatt, D. M.; Meng, E. C. & Ferrin, T. E. UCSF Chimera--a visualization system for exploratory research and analysis (Version 1.15). J Comput Chem. 2004; 25(13): 1605-1612. https://www.ncbi.nlm.nih.gov/pubmed/15264254

Sequence and Features

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
COMPATIBLE WITH RFC[12]
21
COMPATIBLE WITH RFC[21]
23
COMPATIBLE WITH RFC[23]
25
COMPATIBLE WITH RFC[25]
1000
COMPATIBLE WITH RFC[1000]

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 <partinfo>BBa_K3843001 short</partinfo>
-Trypsin is a ubiquitous protease in vertebrates due to its central role in digestion. Trypsin cleaves peptide bonds occurring after positively charged amino acids, such as lysine and arginine. 1UTM, specifically, is a trypsin from Salmo salar (Atlantic salmon). It has a variety of known inhibitors, typically biogenic amines (CITE Ashish's paper). These noncompetitive inhibitors bind to allosteric sites, thereby inactivating trypsin's proteolytic function. Of particular interest to Waterloo iGEM 2021 was the ability of phenylethylamine (PEA), a dopamine precursor, to act as an inhibitor of 1UTM (binding constant CITE). This would allow for 1UTM to be used as a PEA-binding protein. Detection and quantification of phenylethylamine, using 1UTM fused to horseradish peroxidase in a microfluidic flow assay, would give insight into an individual's probability of having ADHD.
+Trypsin is a ubiquitous protease in vertebrates due to its central role in digestion. Trypsin cleaves peptide bonds occurring after positively charged amino acids, such as lysine and arginine. 1UTM (pictured below) is a trypsin from <i>Salmo salar</i> (Atlantic salmon). It has a variety of known inhibitors, typically biogenic amines (Leiros et al., 2009). These noncompetitive inhibitors bind to allosteric sites, thereby inactivating trypsin's proteolytic function. Of particular interest to Waterloo iGEM 2021 was the ability of phenylethylamine (PEA, pictured below in green), a dopamine precursor, to act as an inhibitor of 1UTM, with a dissociation constant Kd = 0.000972 (Leiros et al., 2009; RCSB PDB, n.d.). This would allow for 1UTM to be used as a PEA-binding protein. Detection and quantification of phenylethylamine, using 1UTM fused to horseradish peroxidase in a microfluidic flow assay, would give insight into an individual's probability of having ADHD.
+Waterloo iGEM 2021 sought to improve the binding affinity of 1UTM to PEA in order to increase the reliability of PEA detection in their diagnostic assay. To do so, they employed a computational approach to rational protein design. First, 1UTM was computationally mutated at every residue to every amino acid. Then, the stability of the mutated protein was assessed through overall energy scoring of the lone protein using Rosetta. Next, the protein's binding affinity to phenylethylamine was assessed using ligand docking in AutoDock Vina as well as molecular dynamics simulations in GROMACS, which returned an energy score for the phenylethylamine+1UTM complex; lower energy scores indicated a higher binding affinity. The mutant with the highest binding affinity, where the mutation was in the active site of the protein, was chosen as the improved version of 1UTM. The mutation and evaluation process was repeated until a triple mutant of 1UTM was obtained, containing the mutations Y204F, C195D, and S190T. This affinity-improved version of 1UTM displayed a decrease in the energy scores of the 1UTM-PEA complex by 2.6 kcal/mol (on AutoDock Vina) and 20.8948 kJ/mol (on GROMACS); both correspond to an increase in binding affinity.
+The following image depicts the affinity-improved 1UTM, with 1UTM in orange and PEA in green.
+<html>
+	<div>
+		 <img src="https://2021.igem.org/wiki/images/6/69/T--Waterloo--1UTM%2B.png" alt="">
+	</div>
+</html>
+===References===
+Leiros, H. K. S., Brandsdal, B. O., Andersen, O. A., Os, V., Leiros, I., Helland, R., Otlewski, J., Willassen, N. P., & Smalås, A. O. (2009, January 1). Trypsin specificity as elucidated by lie calculations, x‐ray structures, and association constant measurements. <i>Protein Science, 13</i>(4), 1056-1070. https://onlinelibrary.wiley.com/doi/full/10.1110/ps.03498604.
+Pettersen, E. F.; Goddard, T. D.; Huang, C. C.; Couch, G. S.; Greenblatt, D. M.; Meng, E. C. & Ferrin, T. E. UCSF Chimera--a visualization system for exploratory research and analysis (Version 1.15). <i>J Comput Chem</i>. 2004; <i>25</i>(13): 1605-1612. https://www.ncbi.nlm.nih.gov/pubmed/15264254
-Waterloo iGEM 2021 sought to improve the binding affinity of 1UTM to PEA in order to increase the reliability of PEA detection in their diagnostic assay. To do so, they employed a computational approach to rational protein design. First, 1UTM was computationally mutated at every residue to every amino acid. Then, the stability of the mutated protein was assessed through overall energy scoring of the lone protein using Rosetta. Next, the protein's binding affinity to phenylethylamine was assessed using molecular dynamics simulations in GROMACS, which returned an energy score for the phenylethylamine+1UTM complex; lower energy scores indicated a higher binding affinity. The mutant with the highest binding affinity, where the mutation was in the active site of the protein, was chosen as the improved version of 1UTM. The mutation and evaluation process was repeated once more to obtain a double mutant of 1UTM.