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Construction of chimeric dual-chain avidin by tandem fusion of the related avidins.

Riihimäki TA, Kukkurainen S, Varjonen S, Hörhä J, Nyholm TK, Kulomaa MS, Hytönen VP - PLoS ONE (2011)

Bottom Line: We observed an increase in protein production and better thermal stability, compared with the original dual-chain avidin.The improved dual-chain avidin introduced here increases its potential for future applications.Additionally, this strategy could be helpful when generating hetero-oligomers from other oligomeric proteins with high structural similarity.

View Article: PubMed Central - PubMed

Affiliation: Institute of Biomedical Technology, University of Tampere and Tampere University Hospital, Tampere, Finland.

ABSTRACT

Background: Avidin is a chicken egg-white protein with high affinity to vitamin H, also known as D-biotin. Many applications in life science research are based on this strong interaction. Avidin is a homotetrameric protein, which promotes its modification to symmetrical entities. Dual-chain avidin, a genetically engineered avidin form, has two circularly permuted chicken avidin monomers that are tandem-fused into one polypeptide chain. This form of avidin enables independent modification of the two domains, including the two biotin-binding pockets; however, decreased yields in protein production, compared to wt avidin, and complicated genetic manipulation of two highly similar DNA sequences in the tandem gene have limited the use of dual-chain avidin in biotechnological applications.

Principal findings: To overcome challenges associated with the original dual-chain avidin, we developed chimeric dual-chain avidin, which is a tandem fusion of avidin and avidin-related protein 4 (AVR4), another member of the chicken avidin gene family. We observed an increase in protein production and better thermal stability, compared with the original dual-chain avidin. Additionally, PCR amplification of the hybrid gene was more efficient, thus enabling more convenient and straightforward modification of the dual-chain avidin. When studied closer, the generated chimeric dual-chain avidin showed biphasic biotin dissociation.

Significance: The improved dual-chain avidin introduced here increases its potential for future applications. This molecule offers a valuable base for developing bi-functional avidin tools for bioseparation, carrier proteins, and nanoscale adapters. Additionally, this strategy could be helpful when generating hetero-oligomers from other oligomeric proteins with high structural similarity.

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Related in: MedlinePlus

Interactions between the subunit interfaces of chimeric dcAVD fusions by MD simulation.The electrostatic interaction energy was measured between the circularly permuted subunits in chimeric dcAVDs (A). The analysis was performed for both subunit pairs independently, and the interaction energy is plotted over the 4-ns MD simulation. The van der Waals energy between subunits was measured during the simulation time (B). The potential sources of electrostatic repulsion for dcAVD/SA were examined by visual inspection of the MD simulation data. Three clusters of residues (D247-E15-D241, K12-R239 and D27-E165) potentially causing electrostatic repulsion between cpAVD and cpSA were detected. These residues are shown in a liquorice representation (C, D). The figures were prepared using the program PyMOL (www.pymol.org) and numbered according to Figure 1. All the calculations were performed with 5-ps resolution.
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pone-0020535-g003: Interactions between the subunit interfaces of chimeric dcAVD fusions by MD simulation.The electrostatic interaction energy was measured between the circularly permuted subunits in chimeric dcAVDs (A). The analysis was performed for both subunit pairs independently, and the interaction energy is plotted over the 4-ns MD simulation. The van der Waals energy between subunits was measured during the simulation time (B). The potential sources of electrostatic repulsion for dcAVD/SA were examined by visual inspection of the MD simulation data. Three clusters of residues (D247-E15-D241, K12-R239 and D27-E165) potentially causing electrostatic repulsion between cpAVD and cpSA were detected. These residues are shown in a liquorice representation (C, D). The figures were prepared using the program PyMOL (www.pymol.org) and numbered according to Figure 1. All the calculations were performed with 5-ps resolution.

Mentions: To analyze the possible reasons for the behavioral differences of the chimeric dual-chain avidins, the fusion proteins were modeled based on previously determined 3-D structures of dcAVD (PDB 2C4I), SA (PDB 1MK5), and AVRs (AVR2 (PDB 1WBI), AVR4 (PDB 1Y53)). The model of dcAVD/AVR4 is presented in Figure 1. Molecular dynamics (MD) were performed for the predicted chimeric dual-chain avidin models. The simulations were performed in explicit water using the CHARMM force field. The interaction energy during the MD simulation was measured between subunits, which is the most obvious region in the structure that would cause problems in the dcAVD assembly. In the MD simulation analyses, dcAVD/SA had clearly the least favorable electrostatic interaction energy (Figure 3A), whereas there were no significant differences in the van der Waals energies between chimeric dcAVD forms (Figure 3B). A closer inspection of the MD simulation data of dcAVD/SA revealed three putative electrostatically repulsive interactions (Figures 3C and 3D). These residues (D247-E15-D241, K12-R239 and D27-E165) are potential targets for further engineering of dcAVD/SA to improve its characteristics.


Construction of chimeric dual-chain avidin by tandem fusion of the related avidins.

Riihimäki TA, Kukkurainen S, Varjonen S, Hörhä J, Nyholm TK, Kulomaa MS, Hytönen VP - PLoS ONE (2011)

Interactions between the subunit interfaces of chimeric dcAVD fusions by MD simulation.The electrostatic interaction energy was measured between the circularly permuted subunits in chimeric dcAVDs (A). The analysis was performed for both subunit pairs independently, and the interaction energy is plotted over the 4-ns MD simulation. The van der Waals energy between subunits was measured during the simulation time (B). The potential sources of electrostatic repulsion for dcAVD/SA were examined by visual inspection of the MD simulation data. Three clusters of residues (D247-E15-D241, K12-R239 and D27-E165) potentially causing electrostatic repulsion between cpAVD and cpSA were detected. These residues are shown in a liquorice representation (C, D). The figures were prepared using the program PyMOL (www.pymol.org) and numbered according to Figure 1. All the calculations were performed with 5-ps resolution.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3105096&req=5

pone-0020535-g003: Interactions between the subunit interfaces of chimeric dcAVD fusions by MD simulation.The electrostatic interaction energy was measured between the circularly permuted subunits in chimeric dcAVDs (A). The analysis was performed for both subunit pairs independently, and the interaction energy is plotted over the 4-ns MD simulation. The van der Waals energy between subunits was measured during the simulation time (B). The potential sources of electrostatic repulsion for dcAVD/SA were examined by visual inspection of the MD simulation data. Three clusters of residues (D247-E15-D241, K12-R239 and D27-E165) potentially causing electrostatic repulsion between cpAVD and cpSA were detected. These residues are shown in a liquorice representation (C, D). The figures were prepared using the program PyMOL (www.pymol.org) and numbered according to Figure 1. All the calculations were performed with 5-ps resolution.
Mentions: To analyze the possible reasons for the behavioral differences of the chimeric dual-chain avidins, the fusion proteins were modeled based on previously determined 3-D structures of dcAVD (PDB 2C4I), SA (PDB 1MK5), and AVRs (AVR2 (PDB 1WBI), AVR4 (PDB 1Y53)). The model of dcAVD/AVR4 is presented in Figure 1. Molecular dynamics (MD) were performed for the predicted chimeric dual-chain avidin models. The simulations were performed in explicit water using the CHARMM force field. The interaction energy during the MD simulation was measured between subunits, which is the most obvious region in the structure that would cause problems in the dcAVD assembly. In the MD simulation analyses, dcAVD/SA had clearly the least favorable electrostatic interaction energy (Figure 3A), whereas there were no significant differences in the van der Waals energies between chimeric dcAVD forms (Figure 3B). A closer inspection of the MD simulation data of dcAVD/SA revealed three putative electrostatically repulsive interactions (Figures 3C and 3D). These residues (D247-E15-D241, K12-R239 and D27-E165) are potential targets for further engineering of dcAVD/SA to improve its characteristics.

Bottom Line: We observed an increase in protein production and better thermal stability, compared with the original dual-chain avidin.The improved dual-chain avidin introduced here increases its potential for future applications.Additionally, this strategy could be helpful when generating hetero-oligomers from other oligomeric proteins with high structural similarity.

View Article: PubMed Central - PubMed

Affiliation: Institute of Biomedical Technology, University of Tampere and Tampere University Hospital, Tampere, Finland.

ABSTRACT

Background: Avidin is a chicken egg-white protein with high affinity to vitamin H, also known as D-biotin. Many applications in life science research are based on this strong interaction. Avidin is a homotetrameric protein, which promotes its modification to symmetrical entities. Dual-chain avidin, a genetically engineered avidin form, has two circularly permuted chicken avidin monomers that are tandem-fused into one polypeptide chain. This form of avidin enables independent modification of the two domains, including the two biotin-binding pockets; however, decreased yields in protein production, compared to wt avidin, and complicated genetic manipulation of two highly similar DNA sequences in the tandem gene have limited the use of dual-chain avidin in biotechnological applications.

Principal findings: To overcome challenges associated with the original dual-chain avidin, we developed chimeric dual-chain avidin, which is a tandem fusion of avidin and avidin-related protein 4 (AVR4), another member of the chicken avidin gene family. We observed an increase in protein production and better thermal stability, compared with the original dual-chain avidin. Additionally, PCR amplification of the hybrid gene was more efficient, thus enabling more convenient and straightforward modification of the dual-chain avidin. When studied closer, the generated chimeric dual-chain avidin showed biphasic biotin dissociation.

Significance: The improved dual-chain avidin introduced here increases its potential for future applications. This molecule offers a valuable base for developing bi-functional avidin tools for bioseparation, carrier proteins, and nanoscale adapters. Additionally, this strategy could be helpful when generating hetero-oligomers from other oligomeric proteins with high structural similarity.

Show MeSH
Related in: MedlinePlus