Team:HokkaidoU Japan/Circularization
"
Team:HokkaidoU Japan
\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\
By creating a construction as shown in Fig. 2, the self-assembling region(SAR) and the region containing the SAP interact, thus bringing closer the zip-up linkers on the N-terminal and the C-terminal (Fig. 3).
The zip-up linker plays a vital role in the creation of the covalent bond essential for the circularization of proteins. This consists of amino acid sequence of CWEGGGCGGGCGGGCSALCGGGCGGGCGGG, and is composed of repetition of 3 glycine and 1 cysteine residues. We are hoping that the zip-up linker on the N and C terminal are brought closer by the SAR, and that the cysteine residues form disulfide bonds from the SAR as if to zip up the ends.
Since the distance between the N-terminal and the C-terminal varies depending on the protein, it is essential that a linker of an appropriate length is chosen. This would usually mean that it is necessary to change the linker depending on the protein that is to be circularized. However, our zip-up linker has enough GGGC sequence so that only the flexible part of the linker form disulfide bonds, thus preventing deformation of the tertiary structure. This means that a suitable length of the linker will be used to suit the structure of each protein, enabling this zip-up linker to be applied to various proteins regardless of its structure. Eventually, a structure as indicated in Fig. 4 is obtained.
We designed one construct for circularization of a protein and 3 constructs for negative control. The complete construct is shown in Fig. 5. We chose GFP as the protein part for assay of this biodevice. Two of the negative control construct, ΔSAR and ΔZip, lack SAR or zip-up linker, and hasΔSAR・Zip has only GFP coding sequence with histidine tag, as shown in Fig. 6. We designedΔSAR and ΔZip construct to assess the effect of SAR or zip-up linker. If both parts definitely contribute to efficiency of circularization, the products from these two constructs show less thermostability compared to the products from the complete construct. ΔSAR・Zip construct has only GFP and histidine tag sequence so they wouldn’t show improved thetrmostability.As you can see in the Fig. 5, the complete construct has some restriction enzyme recognition sites, so you can make the three other constructs by digestion with appropriate restriction enzymes.
We ordered DNA sequence of the complete construct from IDT. As shown in Fig. 7, we planned to ligate firstly the complete construct with pSB1C3 vector after PCR and subcloning, and then make three other constructs by digestion with appropriate restriction enzymes.
Add IPTG into cultured bacteria in liquid medium. After confirming expression of GFP, the homogenate which includes GFP will be obtained by causing cell lysis with freeze-thaw. As the products from our 4 DNA constructs have His-tag, you can purify them with Ni-affinity chromatography. After purification of protein, incubate the protein solution under suitable physiochemical conditions to induce self-assembling of the distal SAR of a protein, and then treat with glutathione-S-transeferase to form disulfide bonds between cysteine residues in the zip-up linkers.
First, you need to calculate the concentration of each sample and make it even among 4 samples; product from the complete construct and three negative control constructs. Then treat the protein solution with heat shock at desired tempature for 1 to 5 seconds in PCR cycler. Besides, you need one control sample to treat at 37°C for each construct. After heat shock treatment, cool down the samples on 37°C and compare the fluorescence strength of each sample.
To think about the power of stabilization by circularization, we used HP (Hydrophobic-Polar) model. HP model is a kind of simplified protein folding model and in the model, protein chain is given as zig-zag stick on 2D lattice. Each residue has the characteristic H or P (Hydrophobic or Polar). In this model, if an H residue is next to another H residue except the case it is due to the covalent bond, it decrease free energy because of hydrophobic interaction. In our model, the decreased energy by each hydrophobic interaction is defined as -EH. We added another characteristic SAPs into this model. Through thinking about this model, we can simply think about the effect of SAPs reflected as the effect to possibility to fold into native conformation. We thought SAPs interaction is so strong, so in the case we add SAPs at N terminus and C terminus, both ends are set next to each other in the model. So, let's think about the simplest model.
The simplest model is the model with the number of residue is 4 and the sequence is HPPH. In this case, without SAPs, the number of folding is 5, excluding enantiomers and rotamers. Only the last one is stable and it's energy is -EH. The possibility to fold native conformation is 1×(exp(EH/kBT))/(4×(exp(-0/kBT)+1×(exp(EH/kBT))). To calculate this, we used canonical ensemble from statistical mechanics. The possibility of causing state i is calculated through the function below.
But with SAPs, the number of folding is 1 and the structure is the stablest one. The possibility to fold native conformation is of course 1. Compared with both models, we can obviously think that thanks to the addition of SAPs, we can increase the possibility to fold correctly; the stability of native structure is definitely increased.
Of course, if the native structure's N terminus and C terminus are apart from each other, the addition of SAPs increase the stability of the denatured structure. This means that if we want to stabilize protein of interest with circularization using SAPs, we have to add linkers to make their ends closed.
[1] Scott, C. P., Abel-Santos, E., Wall, M., Wahnon, D. C. & Benkovic, S. J. Production of cyclic peptides and proteins in vivo. Proc. Natl. Acad. Sci. 96, 13638?13643 (1999).
[2] Iwai, H. & Pluckthun, A. Circular beta-lactamase: stability enhancement by cyclizing the backbone. FEBS Lett. 459, 166?72 (1999).
[3] Flory, J. & Yol, S. Theory of Elastic Mechanisms in Fibrous Proteins. 715, 5222?5235 (1956).
[4] Iwai, H., Lingel, a & Pluckthun, a. Cyclic green fluorescent protein produced in vivo using an artificially split PI-PfuI intein from Pyrococcus furiosus. J. Biol. Chem. 276, 16548?54 (2001).
Although enzymes are essential for the development of science, they can only be used under certain conditions due to its nature that it inactivates depending on the temperature and pH. Ways to gain enzymes that are stable against such conditions include investigation of microorganisms that survive under extreme environments, improvement of the activation using amino acid substitution, compartmentalization of the enzymes, and circularization of polypeptides. When enzymes are heated or the pH changed, linear-type enzymes are denatured and deactivated. On the other hand, with circularized enzymes, it is believed that since the ends of the polypeptide are joined and protected, the tertiary structure is less likely to be broken and the activation kept[1][2][3][4]. This year, we attempted in the circularization of proteins using self-assembling peptide(SAP) and zip-up linker.
The SAPs we used are RADA-16-I and P11-4. These are both artificially created amphiphilic SAPs, consisting of amino acid sequence RADARADARADARADA and QQRFEWEFEQQ respectively. They self-assemble under suitable physiochemical conditions due to the polar amino acids and hydrophobic interaction and form β-sheet (Fig. 1).
 |
 |
Fig. 1. RADA16-I and P11-4 self-assemble under suitable physiochemical conditions due to the polar amino acids and hydrophobic interaction and form β-sheet.
By creating a construction as shown in Fig. 2, the self-assembling region(SAR) and the region containing the SAP interact, thus bringing closer the zip-up linkers on the N-terminal and the C-terminal (Fig. 3).
Fig. 2. Genetic construct used for the circularization of proteins using self-assembling peptide(SAP) and zip-up linker
Fig. 3. By using glutathione-S-transeferase, circularization of polypeptides are obtained.
 |
Fig. 4. A structure of circularization of polypeptides |
The zip-up linker plays a vital role in the creation of the covalent bond essential for the circularization of proteins. This consists of amino acid sequence of CWEGGGCGGGCGGGCSALCGGGCGGGCGGG, and is composed of repetition of 3 glycine and 1 cysteine residues. We are hoping that the zip-up linker on the N and C terminal are brought closer by the SAR, and that the cysteine residues form disulfide bonds from the SAR as if to zip up the ends.
Since the distance between the N-terminal and the C-terminal varies depending on the protein, it is essential that a linker of an appropriate length is chosen. This would usually mean that it is necessary to change the linker depending on the protein that is to be circularized. However, our zip-up linker has enough GGGC sequence so that only the flexible part of the linker form disulfide bonds, thus preventing deformation of the tertiary structure. This means that a suitable length of the linker will be used to suit the structure of each protein, enabling this zip-up linker to be applied to various proteins regardless of its structure. Eventually, a structure as indicated in Fig. 4 is obtained.
1. Designing constructs
We designed one construct for circularization of a protein and 3 constructs for negative control. The complete construct is shown in Fig. 5. We chose GFP as the protein part for assay of this biodevice. Two of the negative control construct, ΔSAR and ΔZip, lack SAR or zip-up linker, and hasΔSAR・Zip has only GFP coding sequence with histidine tag, as shown in Fig. 6. We designedΔSAR and ΔZip construct to assess the effect of SAR or zip-up linker. If both parts definitely contribute to efficiency of circularization, the products from these two constructs show less thermostability compared to the products from the complete construct. ΔSAR・Zip construct has only GFP and histidine tag sequence so they wouldn’t show improved thetrmostability.As you can see in the Fig. 5, the complete construct has some restriction enzyme recognition sites, so you can make the three other constructs by digestion with appropriate restriction enzymes.
 |
Fig. 5. The complete construct for protein circularization |
 |
Fig. 6. The constitutions for negative control |
2. Making circuits
We ordered DNA sequence of the complete construct from IDT. As shown in Fig. 7, we planned to ligate firstly the complete construct with pSB1C3 vector after PCR and subcloning, and then make three other constructs by digestion with appropriate restriction enzymes.
 |
Fig. 7. Method for making complete circuit and negative control circuits |
3. IPTG induction, purification and circularization
Add IPTG into cultured bacteria in liquid medium. After confirming expression of GFP, the homogenate which includes GFP will be obtained by causing cell lysis with freeze-thaw. As the products from our 4 DNA constructs have His-tag, you can purify them with Ni-affinity chromatography. After purification of protein, incubate the protein solution under suitable physiochemical conditions to induce self-assembling of the distal SAR of a protein, and then treat with glutathione-S-transeferase to form disulfide bonds between cysteine residues in the zip-up linkers.
 |
Fig. 8. Purification and circularization of protein |
4. Assay
First, you need to calculate the concentration of each sample and make it even among 4 samples; product from the complete construct and three negative control constructs. Then treat the protein solution with heat shock at desired tempature for 1 to 5 seconds in PCR cycler. Besides, you need one control sample to treat at 37°C for each construct. After heat shock treatment, cool down the samples on 37°C and compare the fluorescence strength of each sample.
To think about the power of stabilization by circularization, we used HP (Hydrophobic-Polar) model. HP model is a kind of simplified protein folding model and in the model, protein chain is given as zig-zag stick on 2D lattice. Each residue has the characteristic H or P (Hydrophobic or Polar). In this model, if an H residue is next to another H residue except the case it is due to the covalent bond, it decrease free energy because of hydrophobic interaction. In our model, the decreased energy by each hydrophobic interaction is defined as -EH. We added another characteristic SAPs into this model. Through thinking about this model, we can simply think about the effect of SAPs reflected as the effect to possibility to fold into native conformation. We thought SAPs interaction is so strong, so in the case we add SAPs at N terminus and C terminus, both ends are set next to each other in the model. So, let's think about the simplest model.
The simplest model is the model with the number of residue is 4 and the sequence is HPPH. In this case, without SAPs, the number of folding is 5, excluding enantiomers and rotamers. Only the last one is stable and it's energy is -EH. The possibility to fold native conformation is 1×(exp(EH/kBT))/(4×(exp(-0/kBT)+1×(exp(EH/kBT))). To calculate this, we used canonical ensemble from statistical mechanics. The possibility of causing state i is calculated through the function below.
 |
 |
Fig. 9. ただし配列はHPPHで、できればそれぞれの文字は〇で囲って、それから右端の構造のエネルギーは-E_H |
But with SAPs, the number of folding is 1 and the structure is the stablest one. The possibility to fold native conformation is of course 1. Compared with both models, we can obviously think that thanks to the addition of SAPs, we can increase the possibility to fold correctly; the stability of native structure is definitely increased.
 |
Fig. 10. 図1の状態図の一番右のやつの両末端にSAP(できれば〇で囲って)をつけたものの図 |
Of course, if the native structure's N terminus and C terminus are apart from each other, the addition of SAPs increase the stability of the denatured structure. This means that if we want to stabilize protein of interest with circularization using SAPs, we have to add linkers to make their ends closed.
[1] Scott, C. P., Abel-Santos, E., Wall, M., Wahnon, D. C. & Benkovic, S. J. Production of cyclic peptides and proteins in vivo. Proc. Natl. Acad. Sci. 96, 13638?13643 (1999).
[2] Iwai, H. & Pluckthun, A. Circular beta-lactamase: stability enhancement by cyclizing the backbone. FEBS Lett. 459, 166?72 (1999).
[3] Flory, J. & Yol, S. Theory of Elastic Mechanisms in Fibrous Proteins. 715, 5222?5235 (1956).
[4] Iwai, H., Lingel, a & Pluckthun, a. Cyclic green fluorescent protein produced in vivo using an artificially split PI-PfuI intein from Pyrococcus furiosus. J. Biol. Chem. 276, 16548?54 (2001).
