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| | __NOTOC__ | | __NOTOC__ |
| − | <partinfo>BBa_K3468028 short</partinfo> | + | <partinfo>BBa_K3468068 short</partinfo> |
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| − | The PETase is an enzyme, which can hydrolyze PET and this mutation protein is changed on the basis of the PETase. This protein is changed from A40P,A80V,T113P,T116R,Q119D,Q127L,Q182I,S193R,S214F,R260F,T266P,N275F position which can be more stable in higher temperature compared with the wild type.
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| | <!-- Add more about the biology of this part here | | <!-- Add more about the biology of this part here |
| | ===Usage and Biology=== | | ===Usage and Biology=== |
| | + | The PETase is an enzyme, which can hydrolyze PET and this mutation protein is changed on the basis of the PETase. This protein is changed from S to 46 at D position which can be more stable in higher temperature compared with the wild type. |
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| | <span class='h3bb'>Sequence and Features</span> | | <span class='h3bb'>Sequence and Features</span> |
| − | <partinfo>BBa_K3468028 SequenceAndFeatures</partinfo> | + | <partinfo>BBa_K3468068 SequenceAndFeatures</partinfo> |
| − | The phenyl group of N275F formed a “T-shaped” π-π stacking interaction with the phenyl group of F284.
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| − | Q127L prompts the hydrophobic packing in the protein interior
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| − | A80V prompts the hydrophobic packing in the protein interior.
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| − | The substitution of proline for threonline in T113P mutation reduced the conformational entropy of the local loop region
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| − | The substitution of proline for threonline in T266P mutation reduced the conformational entropy of the local loop region
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| − | For the Q119D mutation, the substitution of asparate for glutamine in Q119D , conferred new hydrogen bonds with the aminogroup of Ser121 and Ser122
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| − | For the T116R mutation, the substitution of arginine for threonine in T116R , conferred new hydrogen bonds with the aminogroup of D118
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| − | For the S193R mutation, the substitution of arginine for serine in S193R , conferred new hydrogen bonds with the carboxyl of A171
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| − | The substitution of proline for alanine in A40P mutation reduced the conformational entropy of the local loop region
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| − | The phenyl group of R260F formed a offset parallel π-π stacking interaction with the phenyl group of F261.
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| − | The phenyl group of S214F formed a offset parallel π-π stacking interaction with the indolyl of the W185.
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| − | Q182I prompts the hydrophobic packing in the protein interior.
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| − | [[File:P40 V80 P113 R116 D119 L127 I182 R193 F214 F260 T266 F275.png|400px]]
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| − | In order to select the mutants with the best thermostability,molecular dynamics (MD) simulation analyses were conducted.In our project, we use GROMACS, which is one of the most popular MD software packages for protein, lipid, and nucleic acid.
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| − | We conducted MD simulations, of wild type PETase (WT) and its eight progressively thermostable mutants performed at 343K.The root mean square deviation (RMSD), the root mean square fluctuation(RMSF), radius of gyration(Rg),solvent accessible surface area(SASA), free-energy landscape and number of hydrogen bond analyses reproduced the correct trends in stability changes of the wild-type and mutated PETase.
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| − | [[File:RNSD-417.png|400px]]
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| − | Many previous studies suggested that proteins with lower RMSD values compared with the starting structure tend to be more thermostable during MD simulation. The RMSD of mutant 417 is lower than the wild-type, and the fluctuation amplitude is smaller, which indicates that mutant 417 is more stable than wild-type PETase at 343K.
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| − | [[File:RNSF-417.png|400px]]
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| − | Mutants with lower RMSF values during MD simulation tend to be more thermostable. RMSF plots of mutant 417 are very similar with wild type PETase and are not indicative of stability at 343K.
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| − | [[File:RG-417 energy.png|400px]]
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| − | The compactness of protein is another indicator to measure the stability of protein. The effect of temperature to the overall dimension of PETaes was gleaned from the Rg analysis. Overall, the Rg of mutant 417 remaines constant throughout the simulation which implies that the mutant structure is capable to maintain its original compactness as the temperature rises.
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| − | [[File:SAS-417.png|400px]]
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| − | Protein folding is driven by hydrophobic effect and is temperature dependent .Under normal conditions, the hydrophilic residues are usually on the protein surface, while hydrophobic residues are generally buried inside the protein away from the aqueous environment. If protein denaturation occurs, the hydrophobic region will be exposed to the solvent.The SASA values of mutant 417 is lower than that of the wild-type, which indicates that mutant 417 is more stable than wild-type PETase at 343K.
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| − | [[File:H-417 energy.png|400px]]
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| − | The hydrogen bond is another important temperature-dependent interaction in maintaining the stability of protein.The higher number of intramolecular hydrogen bonds conferred mutants have higher resistant against heat denaturation. The number of intramolecular hydrogen bonds in mutant 417 is very similar with wild type PETase.
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| − | [[File:417 energy.png|400px]]
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| − | The mutant 417 shows the required flexibility at appropriate temperature ranges and maintain conformational stability at high temperature. It shows a deep and rugged free-energy landscape,which indicates mutant 417 has more possibilities to have higher thermostability.
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| − | Docing Results;
| + | This mutation increases the hydrogen bond near S46. The hydrogen bond is an interaction formed by N, O and H in a protein, which can stabilize the structure of the protein and improve thermal stability. |
| − | The benzene ring is located in a gully between two aromatic residues TYR87 and TRP185.
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| − | The distance of PI-PI bond is 5.0A between TRP185 and the benzene ring in part of the first MHET. | + | |
| − | The whole ligand just fits into the pocket and binds tightly.
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| − | Three residues of Ser160, Asp206 and His237 formed a catalytic triplet, and ranging was performed: Y87:4.7A ;M161:5.4A; H237:4.8A.
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| − | Significance of molecular docking for mutant work:
| + | We use FoldX to judge the thermal stability of S46D and Jmol to find whether there is a new salt bridge. Finally we use Pymol to find out what interaction make it become stable. |
| − | The molecular docking model can predict the binding mode and affinity of mutants and ligands, and promote the work of mutants by analyzing the result model.
| + | [[File:PETase A40P,A80V,T113P,T116R,Q119D,Q127L,Q182I,S193R,S214F,R260F,T266P,N275F.jpeg|400px|thumb|left|Fig.1 PETase A40P,A80V,T113P,T116R,Q119D,Q127L,Q182I,S193R,S214F,R260F,T266P,N275F in docking]] |
| | <!-- Uncomment this to enable Functional Parameter display | | <!-- Uncomment this to enable Functional Parameter display |
| | ===Functional Parameters=== | | ===Functional Parameters=== |
| − | <partinfo>BBa_K3468028 parameters</partinfo> | + | <partinfo>BBa_K3468068 parameters</partinfo> |
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This mutation increases the hydrogen bond near S46. The hydrogen bond is an interaction formed by N, O and H in a protein, which can stabilize the structure of the protein and improve thermal stability.
We use FoldX to judge the thermal stability of S46D and Jmol to find whether there is a new salt bridge. Finally we use Pymol to find out what interaction make it become stable.
