Limits...
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.

Show MeSH

Related in: MedlinePlus

Local dynamics in the chimeric dcAVD fusions measured by MD simulation.To probe the local structural dynamics, the root mean square fluctuation (RMSF) per residue was measured for a short time window (10 ps) for the last 3 ns of the 4-ns MD simulation. The resulting values were averaged and plotted in graphs A–C (A: dcAVD/AVR2; B: dcAVD/AVR4; C: dcAVD/SA). The dynamics of the structure are illustrated by plotting 50 superimposed structural snapshots along the 4-ns simulation (D–F). The loops showing a high amount of structural fluctuation (RMSF>1.5 Å) are indicated by numbers referring to the amino acid sequence (see also Figure 1). The structural snapshots are colored according to timestep, as illustrated by the scale bar (please note that the color scale is illustrative only because of the rendering method). Figures D–F were prepared using the program VMD 1.8.7 [35].
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3105096&req=5

pone-0020535-g004: Local dynamics in the chimeric dcAVD fusions measured by MD simulation.To probe the local structural dynamics, the root mean square fluctuation (RMSF) per residue was measured for a short time window (10 ps) for the last 3 ns of the 4-ns MD simulation. The resulting values were averaged and plotted in graphs A–C (A: dcAVD/AVR2; B: dcAVD/AVR4; C: dcAVD/SA). The dynamics of the structure are illustrated by plotting 50 superimposed structural snapshots along the 4-ns simulation (D–F). The loops showing a high amount of structural fluctuation (RMSF>1.5 Å) are indicated by numbers referring to the amino acid sequence (see also Figure 1). The structural snapshots are colored according to timestep, as illustrated by the scale bar (please note that the color scale is illustrative only because of the rendering method). Figures D–F were prepared using the program VMD 1.8.7 [35].

Mentions: To investigate how the chimeric dcAVD forms differ in terms of loop dynamics in MD simulations, we analyzed a root mean square fluctuation (RMSF), measuring main chain motion for a 10 ps time window (Figure 4). The analysis revealed that the linkers connecting the original termini of the domains (Figure 1, red linkers) were highly mobile, particularly in the avidin domain (GGSGGS, residues 70–75 connecting the original termini). We also detected regions behaving differently between the chimeric dcAVD forms; for dcAVD/SA, there were unique mobile regions in the loop between the strands β7 and β8 (residues 164–169) and in the loop between the strands β4 and β5 (residues 243–249) that corresponded to the potential electrostatic repulsion (Figure 3C and 3D). Overall, however, no dramatic differences were observed in the loop mobility between different dcAVD forms, suggesting that there were no significant problems in the molecular design of the chimeric dcAVDs.


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)

Local dynamics in the chimeric dcAVD fusions measured by MD simulation.To probe the local structural dynamics, the root mean square fluctuation (RMSF) per residue was measured for a short time window (10 ps) for the last 3 ns of the 4-ns MD simulation. The resulting values were averaged and plotted in graphs A–C (A: dcAVD/AVR2; B: dcAVD/AVR4; C: dcAVD/SA). The dynamics of the structure are illustrated by plotting 50 superimposed structural snapshots along the 4-ns simulation (D–F). The loops showing a high amount of structural fluctuation (RMSF>1.5 Å) are indicated by numbers referring to the amino acid sequence (see also Figure 1). The structural snapshots are colored according to timestep, as illustrated by the scale bar (please note that the color scale is illustrative only because of the rendering method). Figures D–F were prepared using the program VMD 1.8.7 [35].
© Copyright Policy
Related In: Results  -  Collection

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

pone-0020535-g004: Local dynamics in the chimeric dcAVD fusions measured by MD simulation.To probe the local structural dynamics, the root mean square fluctuation (RMSF) per residue was measured for a short time window (10 ps) for the last 3 ns of the 4-ns MD simulation. The resulting values were averaged and plotted in graphs A–C (A: dcAVD/AVR2; B: dcAVD/AVR4; C: dcAVD/SA). The dynamics of the structure are illustrated by plotting 50 superimposed structural snapshots along the 4-ns simulation (D–F). The loops showing a high amount of structural fluctuation (RMSF>1.5 Å) are indicated by numbers referring to the amino acid sequence (see also Figure 1). The structural snapshots are colored according to timestep, as illustrated by the scale bar (please note that the color scale is illustrative only because of the rendering method). Figures D–F were prepared using the program VMD 1.8.7 [35].
Mentions: To investigate how the chimeric dcAVD forms differ in terms of loop dynamics in MD simulations, we analyzed a root mean square fluctuation (RMSF), measuring main chain motion for a 10 ps time window (Figure 4). The analysis revealed that the linkers connecting the original termini of the domains (Figure 1, red linkers) were highly mobile, particularly in the avidin domain (GGSGGS, residues 70–75 connecting the original termini). We also detected regions behaving differently between the chimeric dcAVD forms; for dcAVD/SA, there were unique mobile regions in the loop between the strands β7 and β8 (residues 164–169) and in the loop between the strands β4 and β5 (residues 243–249) that corresponded to the potential electrostatic repulsion (Figure 3C and 3D). Overall, however, no dramatic differences were observed in the loop mobility between different dcAVD forms, suggesting that there were no significant problems in the molecular design of the chimeric dcAVDs.

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