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Markku S. Kulomaa - One of the best experts on this subject based on the ideXlab platform.
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Construction of chimeric dual-chain Avidin by tandem fusion of the related Avidins.
PloS one, 2011Co-Authors: Tiina A. Riihimäki, Markku S. Kulomaa, Thomas K M Nyholm, Sampo Kukkurainen, Jarno Hörhä, Suvi Varjonen, Vesa P HytonenAbstract: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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Structural and functional characteristics of xenAvidin, the first frog Avidin from Xenopus tropicalis
BMC Structural Biology, 2009Co-Authors: Juha A E Maatta, Markku S. Kulomaa, Vesa P Hytonen, Satu H Helppolainen, Mark S Johnson, Tomi T Airenne, Henri R NordlundAbstract:Background Avidins are proteins with extraordinarily high ligand-binding affinity, a property which is used in a wide array of life science applications. Even though useful for biotechnology and nanotechnology, the biological function of Avidins is not fully understood. Here we structurally and functionally characterise a novel Avidin named xenAvidin, which is to our knowledge the first reported Avidin from a frog. Results XenAvidin was identified from an EST sequence database for Xenopus tropicalis and produced in insect cells using a baculovirus expression system. The recombinant xenAvidin was found to be homotetrameric based on gel filtration analysis. Biacore sensor analysis, fluorescently labelled biotin and radioactive biotin were used to evaluate the biotin-binding properties of xenAvidin - it binds biotin with high affinity though less tightly than do chicken Avidin and bacterial streptAvidin. X-ray crystallography revealed structural conservation around the ligand-binding site, while some of the loop regions have a unique design. The location of structural water molecules at the entrance and/or within the ligand-binding site may have a role in determining the characteristic biotin-binding properties of xenAvidin. Conclusion The novel data reported here provide information about the biochemically and structurally important determinants of biotin binding. This information may facilitate the discovery of novel tools for biotechnology.
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crystal structure of rhizAvidin insights into the enigmatic high affinity interaction of an innate biotin binding protein dimer
Journal of Molecular Biology, 2009Co-Authors: Amit Meir, Markku S. Kulomaa, Vesa P Hytonen, Henri R Nordlund, Meir Wilchek, Satu H Helppolainen, Erez Podoly, Juha A E Maatta, Edward A Bayer, Oded LivnahAbstract:Abstract RhizAvidin, from the proteobacterium Rhizobium etli, exhibits high affinity towards biotin but maintains an inherent dimeric quaternary structure and thus, differs from all other known tetrameric Avidins. RhizAvidin also differs from the other Avidins, since it lacks the characteristic tryptophan residue positioned in the L7,8 loop that plays a crucial role in high-affinity binding and oligomeric stability of the tetrameric Avidins. The question is, therefore, how does the dimer exist and how is the high biotin-binding affinity retained? For this purpose, the crystal structures of apo- and biotin-complexed rhizAvidin were determined. The structures reveal that the rhizAvidin monomer exhibits a topology similar to those of other members of the Avidin family, that is, eight antiparallel β-strands that form the conventional Avidin β-barrel. The quaternary structure comprises the sandwich-like dimer, in which the extensive 1–4 intermonomer interface is intact, but the 1–2 and 1–3 interfaces are nonexistent. Consequently, the biotin-binding site is partially accessible, due to the lack of the tryptophan “lid” that distinguishes the tetrameric structures. In rhizAvidin, a disulfide bridge connecting the L3,4 and L5,6 loops restrains the L3,4 loop conformation, leaving the binding-site residues essentially unchanged upon biotin binding. Our study suggests that in addition to the characteristic hydrogen bonding and hydrophobic interactions, the preformed architecture of the binding site and consequent shape complementarity play a decisive role in the high-affinity biotin binding of rhizAvidin. The structural description of a novel dimeric Avidin-like molecule will greatly contribute to the design of improved and unique Avidin derivatives for diversifying the capabilities of Avidin–biotin technology.
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Brave new (strept)Avidins in biotechnology
Trends in biotechnology, 2007Co-Authors: Olli H Laitinen, Vesa P Hytonen, Henri R Nordlund, Markku S. KulomaaAbstract:Avidin and streptAvidin are widely used in (strept)Avidin–biotin technology, which is based on their tight biotin-binding capability. These techniques are exceptionally diverse, ranging from simple purification and labeling methods to sophisticated drug pre-targeting and nanostructure-building approaches. Improvements in protein engineering have provided new possibilities to develop tailored protein tools. The (strept)Avidin scaffold has been engineered to extend the existing range of applications and to develop new ones. Modifications to (strept)Avidins – such as simple amino acid substitutions to reduce biotin binding and alter physico–chemical characters – have recently developed into more sophisticated changes, including chimeric (strept)Avidins, topology rearrangements and stitching of non-natural amino acids into the active sites. In this review, we highlight the current status in genetically engineered (strept)Avidins and illustrate their versatility as advanced tools in the multiple fields of modern bioscience, medicine and nanotechnology.
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Genetically engineered Avidins and streptAvidins.
Cellular and molecular life sciences : CMLS, 2006Co-Authors: Olli H Laitinen, Vesa P Hytonen, Henri R Nordlund, Markku S. KulomaaAbstract:Chicken Avidin and bacterial streptAvidin, (strept)Avidin, are proteins widely utilized in a number of applications in life science, ranging from purification and labeling techniques to diagnostics, and from targeted drug delivery to nanotechnology. (Strept)Avidin-biotin technology relies on the extremely tight and specific affinity between (strept)Avidin and biotin (dissociation constant, Kd≈10−14–10−16 M). (Strept)Avidins are also exceptionally stable proteins. To study their ligand binding and stability characteristics, the two proteins have been extensively modified both chemically and genetically. There are excellent accounts of this technology and chemically modified (strept)Avidins, but no comprehensive reviews exist concerning genetically engineered (strept)Avidins. To fill this gap, we here go through the genetically engineered (strept)Avidins, summarizing how these constructs were designed and how they have improved our understanding of the structural and functional characteristics of these proteins, and the benefits they have provided for (strept)Avidin-biotin technology.
Vesa P Hytonen - One of the best experts on this subject based on the ideXlab platform.
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Bacterial Avidins are a widely distributed protein family in Actinobacteria, Proteobacteria and Bacteroidetes
BMC Ecology and Evolution, 2021Co-Authors: Olli H Laitinen, Tanja P. Kuusela, Sampo Kukkurainen, Anssi Nurminen, Aki Sinkkonen, Vesa P HytonenAbstract:Background Avidins are biotin-binding proteins commonly found in the vertebrate eggs. In addition to streptAvidin from Streptomyces Avidinii , a growing number of Avidins have been characterized from divergent bacterial species. However, a systematic research concerning their taxonomy and ecological role has never been done. We performed a search for Avidin encoding genes among bacteria using available databases and classified potential Avidins according to taxonomy and the ecological niches utilized by host bacteria. Results Numerous Avidin-encoding genes were found in the phyla Actinobacteria and Proteobacteria. The diversity of protein sequences was high and several new variants of genes encoding biotin-binding Avidins were found. The living strategies of bacteria hosting Avidin encoding genes fall mainly into two categories. Human and animal pathogens were overrepresented among the found bacteria carrying Avidin genes. The other widespread category were bacteria that either fix nitrogen or live in root nodules/rhizospheres of plants hosting nitrogen-fixing bacteria. Conclusions Bacterial Avidins are a taxonomically and ecologically diverse group mainly found in Actinobacteria, Proteobacteria and Bacteroidetes, associated often with plant invasiveness. Avidin encoding genes in plasmids hint that Avidins may be horizontally transferred. The current survey may be used as a basis in attempts to understand the ecological significance of biotin-binding capacity.
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Structural and Functional Characteristics of Chimeric Avidins Physically Adsorbed onto Functionalized Polythiophene Thin Films
ACS Applied Materials & Interfaces, 2012Co-Authors: Willem M Albers, Vesa P Hytonen, Juha A E Maatta, Jani M Pelto, Clément Suspène, Abderrahim Yassar, Inger M Vikholm-lundin, Kirsi TappuraAbstract:Stabilized bioreceptor layers are of great importance in the design of novel biosensors. In earlier work, chimeric Avidins enabled immobilization of biotinylated antibodies onto gold surfaces with greater stability compared to more conventional Avidins (wild-type Avidin and streptAvidin). In the present study, the applicability of chimeric Avidins as a general binding scaffold for biotinylated antibodies on spin-coated functionalized polythiophene thin films has been studied by surface plasmon resonance and atomic force microscopy. Novel chimeric Avidins showed remarkably increased binding characteristics compared with other Avidins, such as wild-type Avidin, streptAvidin, and bacterial Avidin when merely physically adsorbed onto the polythiophene surface. They gave the highest binding capacities, the highest affinity constant, and the highest stability for biotinylated probe immobilization. Introduction of carboxylic acid groups to polythiophene layer further enhanced the binding level of the Avidins. Polythiophene layers functionalized with chimeric Avidins thus offered a promising generic platform for biosensor applications.
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Construction of chimeric dual-chain Avidin by tandem fusion of the related Avidins.
PloS one, 2011Co-Authors: Tiina A. Riihimäki, Markku S. Kulomaa, Thomas K M Nyholm, Sampo Kukkurainen, Jarno Hörhä, Suvi Varjonen, Vesa P HytonenAbstract: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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Structural and functional characteristics of xenAvidin, the first frog Avidin from Xenopus tropicalis
BMC Structural Biology, 2009Co-Authors: Juha A E Maatta, Markku S. Kulomaa, Vesa P Hytonen, Satu H Helppolainen, Mark S Johnson, Tomi T Airenne, Henri R NordlundAbstract:Background Avidins are proteins with extraordinarily high ligand-binding affinity, a property which is used in a wide array of life science applications. Even though useful for biotechnology and nanotechnology, the biological function of Avidins is not fully understood. Here we structurally and functionally characterise a novel Avidin named xenAvidin, which is to our knowledge the first reported Avidin from a frog. Results XenAvidin was identified from an EST sequence database for Xenopus tropicalis and produced in insect cells using a baculovirus expression system. The recombinant xenAvidin was found to be homotetrameric based on gel filtration analysis. Biacore sensor analysis, fluorescently labelled biotin and radioactive biotin were used to evaluate the biotin-binding properties of xenAvidin - it binds biotin with high affinity though less tightly than do chicken Avidin and bacterial streptAvidin. X-ray crystallography revealed structural conservation around the ligand-binding site, while some of the loop regions have a unique design. The location of structural water molecules at the entrance and/or within the ligand-binding site may have a role in determining the characteristic biotin-binding properties of xenAvidin. Conclusion The novel data reported here provide information about the biochemically and structurally important determinants of biotin binding. This information may facilitate the discovery of novel tools for biotechnology.
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crystal structure of rhizAvidin insights into the enigmatic high affinity interaction of an innate biotin binding protein dimer
Journal of Molecular Biology, 2009Co-Authors: Amit Meir, Markku S. Kulomaa, Vesa P Hytonen, Henri R Nordlund, Meir Wilchek, Satu H Helppolainen, Erez Podoly, Juha A E Maatta, Edward A Bayer, Oded LivnahAbstract:Abstract RhizAvidin, from the proteobacterium Rhizobium etli, exhibits high affinity towards biotin but maintains an inherent dimeric quaternary structure and thus, differs from all other known tetrameric Avidins. RhizAvidin also differs from the other Avidins, since it lacks the characteristic tryptophan residue positioned in the L7,8 loop that plays a crucial role in high-affinity binding and oligomeric stability of the tetrameric Avidins. The question is, therefore, how does the dimer exist and how is the high biotin-binding affinity retained? For this purpose, the crystal structures of apo- and biotin-complexed rhizAvidin were determined. The structures reveal that the rhizAvidin monomer exhibits a topology similar to those of other members of the Avidin family, that is, eight antiparallel β-strands that form the conventional Avidin β-barrel. The quaternary structure comprises the sandwich-like dimer, in which the extensive 1–4 intermonomer interface is intact, but the 1–2 and 1–3 interfaces are nonexistent. Consequently, the biotin-binding site is partially accessible, due to the lack of the tryptophan “lid” that distinguishes the tetrameric structures. In rhizAvidin, a disulfide bridge connecting the L3,4 and L5,6 loops restrains the L3,4 loop conformation, leaving the binding-site residues essentially unchanged upon biotin binding. Our study suggests that in addition to the characteristic hydrogen bonding and hydrophobic interactions, the preformed architecture of the binding site and consequent shape complementarity play a decisive role in the high-affinity biotin binding of rhizAvidin. The structural description of a novel dimeric Avidin-like molecule will greatly contribute to the design of improved and unique Avidin derivatives for diversifying the capabilities of Avidin–biotin technology.
Henri R Nordlund - One of the best experts on this subject based on the ideXlab platform.
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Structural and functional characteristics of xenAvidin, the first frog Avidin from Xenopus tropicalis
BMC Structural Biology, 2009Co-Authors: Juha A E Maatta, Markku S. Kulomaa, Vesa P Hytonen, Satu H Helppolainen, Mark S Johnson, Tomi T Airenne, Henri R NordlundAbstract:Background Avidins are proteins with extraordinarily high ligand-binding affinity, a property which is used in a wide array of life science applications. Even though useful for biotechnology and nanotechnology, the biological function of Avidins is not fully understood. Here we structurally and functionally characterise a novel Avidin named xenAvidin, which is to our knowledge the first reported Avidin from a frog. Results XenAvidin was identified from an EST sequence database for Xenopus tropicalis and produced in insect cells using a baculovirus expression system. The recombinant xenAvidin was found to be homotetrameric based on gel filtration analysis. Biacore sensor analysis, fluorescently labelled biotin and radioactive biotin were used to evaluate the biotin-binding properties of xenAvidin - it binds biotin with high affinity though less tightly than do chicken Avidin and bacterial streptAvidin. X-ray crystallography revealed structural conservation around the ligand-binding site, while some of the loop regions have a unique design. The location of structural water molecules at the entrance and/or within the ligand-binding site may have a role in determining the characteristic biotin-binding properties of xenAvidin. Conclusion The novel data reported here provide information about the biochemically and structurally important determinants of biotin binding. This information may facilitate the discovery of novel tools for biotechnology.
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crystal structure of rhizAvidin insights into the enigmatic high affinity interaction of an innate biotin binding protein dimer
Journal of Molecular Biology, 2009Co-Authors: Amit Meir, Markku S. Kulomaa, Vesa P Hytonen, Henri R Nordlund, Meir Wilchek, Satu H Helppolainen, Erez Podoly, Juha A E Maatta, Edward A Bayer, Oded LivnahAbstract:Abstract RhizAvidin, from the proteobacterium Rhizobium etli, exhibits high affinity towards biotin but maintains an inherent dimeric quaternary structure and thus, differs from all other known tetrameric Avidins. RhizAvidin also differs from the other Avidins, since it lacks the characteristic tryptophan residue positioned in the L7,8 loop that plays a crucial role in high-affinity binding and oligomeric stability of the tetrameric Avidins. The question is, therefore, how does the dimer exist and how is the high biotin-binding affinity retained? For this purpose, the crystal structures of apo- and biotin-complexed rhizAvidin were determined. The structures reveal that the rhizAvidin monomer exhibits a topology similar to those of other members of the Avidin family, that is, eight antiparallel β-strands that form the conventional Avidin β-barrel. The quaternary structure comprises the sandwich-like dimer, in which the extensive 1–4 intermonomer interface is intact, but the 1–2 and 1–3 interfaces are nonexistent. Consequently, the biotin-binding site is partially accessible, due to the lack of the tryptophan “lid” that distinguishes the tetrameric structures. In rhizAvidin, a disulfide bridge connecting the L3,4 and L5,6 loops restrains the L3,4 loop conformation, leaving the binding-site residues essentially unchanged upon biotin binding. Our study suggests that in addition to the characteristic hydrogen bonding and hydrophobic interactions, the preformed architecture of the binding site and consequent shape complementarity play a decisive role in the high-affinity biotin binding of rhizAvidin. The structural description of a novel dimeric Avidin-like molecule will greatly contribute to the design of improved and unique Avidin derivatives for diversifying the capabilities of Avidin–biotin technology.
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Brave new (strept)Avidins in biotechnology
Trends in biotechnology, 2007Co-Authors: Olli H Laitinen, Vesa P Hytonen, Henri R Nordlund, Markku S. KulomaaAbstract:Avidin and streptAvidin are widely used in (strept)Avidin–biotin technology, which is based on their tight biotin-binding capability. These techniques are exceptionally diverse, ranging from simple purification and labeling methods to sophisticated drug pre-targeting and nanostructure-building approaches. Improvements in protein engineering have provided new possibilities to develop tailored protein tools. The (strept)Avidin scaffold has been engineered to extend the existing range of applications and to develop new ones. Modifications to (strept)Avidins – such as simple amino acid substitutions to reduce biotin binding and alter physico–chemical characters – have recently developed into more sophisticated changes, including chimeric (strept)Avidins, topology rearrangements and stitching of non-natural amino acids into the active sites. In this review, we highlight the current status in genetically engineered (strept)Avidins and illustrate their versatility as advanced tools in the multiple fields of modern bioscience, medicine and nanotechnology.
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Genetically engineered Avidins and streptAvidins.
Cellular and molecular life sciences : CMLS, 2006Co-Authors: Olli H Laitinen, Vesa P Hytonen, Henri R Nordlund, Markku S. KulomaaAbstract:Chicken Avidin and bacterial streptAvidin, (strept)Avidin, are proteins widely utilized in a number of applications in life science, ranging from purification and labeling techniques to diagnostics, and from targeted drug delivery to nanotechnology. (Strept)Avidin-biotin technology relies on the extremely tight and specific affinity between (strept)Avidin and biotin (dissociation constant, Kd≈10−14–10−16 M). (Strept)Avidins are also exceptionally stable proteins. To study their ligand binding and stability characteristics, the two proteins have been extensively modified both chemically and genetically. There are excellent accounts of this technology and chemically modified (strept)Avidins, but no comprehensive reviews exist concerning genetically engineered (strept)Avidins. To fill this gap, we here go through the genetically engineered (strept)Avidins, summarizing how these constructs were designed and how they have improved our understanding of the structural and functional characteristics of these proteins, and the benefits they have provided for (strept)Avidin-biotin technology.
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Tetravalent single-chain Avidin: from subunits to protein domains via circularly permuted Avidins.
Biochemical Journal, 2005Co-Authors: Henri R Nordlund, Vesa P Hytonen, Olli H Laitinen, Juha A E Maatta, Jarno Hörhä, Eevaleena J. Porkka, Daniel J. White, Katrin K. Halling, J. Peter Slotte, Markku S. KulomaaAbstract:scAvd (single-chain Avidin, where two dcAvd are joined in a single polypeptide chain), having four biotin-binding domains, was constructed by fusion of topologically modified Avidin units. scAvd showed similar biotin binding and thermal stability properties as chicken Avidin. The DNA construct encoding scAvd contains four circularly permuted Avidin domains, plus short linkers connecting the four domains into a single polypeptide chain. In contrast with wild-type Avidin, which contains four identical Avidin monomers, scAvd enables each one of the four Avidin domains to be independently modified by protein engineering. Therefore the scAvd scaffold can be used to construct spatially and stoichiometrically defined pseudotetrameric Avidin molecules showing different domain characteristics. In addition, unmodified scAvd could be used as a fusion partner, since it provides a unique non-oligomeric structure, which is fully functional with four high-affinity biotin-binding sites. Furthermore, the subunit-to-domain strategy described in the present study could be applied to other proteins and protein complexes, facilitating the development of sophisticated protein tools for applications in nanotechnology and life sciences.
Juha A E Maatta - One of the best experts on this subject based on the ideXlab platform.
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Structural and Functional Characteristics of Chimeric Avidins Physically Adsorbed onto Functionalized Polythiophene Thin Films
ACS Applied Materials & Interfaces, 2012Co-Authors: Willem M Albers, Vesa P Hytonen, Juha A E Maatta, Jani M Pelto, Clément Suspène, Abderrahim Yassar, Inger M Vikholm-lundin, Kirsi TappuraAbstract:Stabilized bioreceptor layers are of great importance in the design of novel biosensors. In earlier work, chimeric Avidins enabled immobilization of biotinylated antibodies onto gold surfaces with greater stability compared to more conventional Avidins (wild-type Avidin and streptAvidin). In the present study, the applicability of chimeric Avidins as a general binding scaffold for biotinylated antibodies on spin-coated functionalized polythiophene thin films has been studied by surface plasmon resonance and atomic force microscopy. Novel chimeric Avidins showed remarkably increased binding characteristics compared with other Avidins, such as wild-type Avidin, streptAvidin, and bacterial Avidin when merely physically adsorbed onto the polythiophene surface. They gave the highest binding capacities, the highest affinity constant, and the highest stability for biotinylated probe immobilization. Introduction of carboxylic acid groups to polythiophene layer further enhanced the binding level of the Avidins. Polythiophene layers functionalized with chimeric Avidins thus offered a promising generic platform for biosensor applications.
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Structural and functional characteristics of xenAvidin, the first frog Avidin from Xenopus tropicalis
BMC Structural Biology, 2009Co-Authors: Juha A E Maatta, Markku S. Kulomaa, Vesa P Hytonen, Satu H Helppolainen, Mark S Johnson, Tomi T Airenne, Henri R NordlundAbstract:Background Avidins are proteins with extraordinarily high ligand-binding affinity, a property which is used in a wide array of life science applications. Even though useful for biotechnology and nanotechnology, the biological function of Avidins is not fully understood. Here we structurally and functionally characterise a novel Avidin named xenAvidin, which is to our knowledge the first reported Avidin from a frog. Results XenAvidin was identified from an EST sequence database for Xenopus tropicalis and produced in insect cells using a baculovirus expression system. The recombinant xenAvidin was found to be homotetrameric based on gel filtration analysis. Biacore sensor analysis, fluorescently labelled biotin and radioactive biotin were used to evaluate the biotin-binding properties of xenAvidin - it binds biotin with high affinity though less tightly than do chicken Avidin and bacterial streptAvidin. X-ray crystallography revealed structural conservation around the ligand-binding site, while some of the loop regions have a unique design. The location of structural water molecules at the entrance and/or within the ligand-binding site may have a role in determining the characteristic biotin-binding properties of xenAvidin. Conclusion The novel data reported here provide information about the biochemically and structurally important determinants of biotin binding. This information may facilitate the discovery of novel tools for biotechnology.
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crystal structure of rhizAvidin insights into the enigmatic high affinity interaction of an innate biotin binding protein dimer
Journal of Molecular Biology, 2009Co-Authors: Amit Meir, Markku S. Kulomaa, Vesa P Hytonen, Henri R Nordlund, Meir Wilchek, Satu H Helppolainen, Erez Podoly, Juha A E Maatta, Edward A Bayer, Oded LivnahAbstract:Abstract RhizAvidin, from the proteobacterium Rhizobium etli, exhibits high affinity towards biotin but maintains an inherent dimeric quaternary structure and thus, differs from all other known tetrameric Avidins. RhizAvidin also differs from the other Avidins, since it lacks the characteristic tryptophan residue positioned in the L7,8 loop that plays a crucial role in high-affinity binding and oligomeric stability of the tetrameric Avidins. The question is, therefore, how does the dimer exist and how is the high biotin-binding affinity retained? For this purpose, the crystal structures of apo- and biotin-complexed rhizAvidin were determined. The structures reveal that the rhizAvidin monomer exhibits a topology similar to those of other members of the Avidin family, that is, eight antiparallel β-strands that form the conventional Avidin β-barrel. The quaternary structure comprises the sandwich-like dimer, in which the extensive 1–4 intermonomer interface is intact, but the 1–2 and 1–3 interfaces are nonexistent. Consequently, the biotin-binding site is partially accessible, due to the lack of the tryptophan “lid” that distinguishes the tetrameric structures. In rhizAvidin, a disulfide bridge connecting the L3,4 and L5,6 loops restrains the L3,4 loop conformation, leaving the binding-site residues essentially unchanged upon biotin binding. Our study suggests that in addition to the characteristic hydrogen bonding and hydrophobic interactions, the preformed architecture of the binding site and consequent shape complementarity play a decisive role in the high-affinity biotin binding of rhizAvidin. The structural description of a novel dimeric Avidin-like molecule will greatly contribute to the design of improved and unique Avidin derivatives for diversifying the capabilities of Avidin–biotin technology.
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rhizAvidin from rhizobium etli the first natural dimer in the Avidin protein family
Biochemical Journal, 2007Co-Authors: Satu H Helppolainen, Juha A E Maatta, Katrin K. Halling, Kari J. Airenne, Tuulia Huhtala, Timo Liimatainen, Kirsi Nurminen, Peter J Slotte, Seppo Ylaherttuala, Ale NärvänenAbstract:Rhizobium etli CFN42 is a symbiotic nitrogen-fixing bacterium of the common bean Phaseolus vulgaris. The symbiotic plasmid p42d of R. etli comprises a gene encoding a putative (strept)Avidin-like protein, named rhizAvidin. The amino acid sequence identity of rhizAvidin in relation to other known Avidin-like proteins is 20–30%. The amino acid residues involved in the (strept)Avidin–biotin interaction are well conserved in rhizAvidin. The structural and functional properties of rhizAvidin were carefully studied, and we found that rhizAvidin shares characteristics with bradAvidin, streptAvidin and Avidin. However, we found that it is the first naturally occurring dimeric protein in the Avidin protein family, in contrast with tetrameric (strept)Avidin and bradAvidin. Moreover, it possesses a proline residue after a flexible loop (GGSG) in a position close to Trp-110 in Avidin, which is an important biotin-binding residue. [3H]Biotin dissociation and ITC (isothermal titration calorimetry) experiments showed dimeric rhizAvidin to be a high-affinity biotin-binding protein. Its thermal stability was lower than that of Avidin; although similar to streptAvidin, it was insensitive to proteinase K. The immunological cross-reactivity of rhizAvidin was tested with human serum samples obtained from cancer patients exposed to (strept)Avidin. No significant cross-reactivity was observed. The biodistribution of the protein was studied by SPECT (single-photon emission computed tomography) imaging in rats. Similarly to Avidin, rhizAvidin was observed to accumulate rapidly, mainly in the liver. Evidently, rhizAvidin could be used as a complement to (strept)Avidin in (strept)Avidin–biotin technology.
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RhizAvidin from Rhizobium etli: the first natural dimer in the Avidin protein family
Biochemical Journal, 2007Co-Authors: Satu H Helppolainen, Juha A E Maatta, Katrin K. Halling, J. Peter Slotte, Kari J. Airenne, Kirsi P Nurminen, Tuulia Huhtala, Timo Liimatainen, Seppo Ylä-herttuala, Ale NärvänenAbstract:Rhizobium etli CFN42 is a symbiotic, nitrogen-fixing bacterium of the common bean, Phaseolus vulgaris. The symbiotic plasmid p42d of R. etli comprises a gene encoding a putative (strept)Avidin-like protein, named rhizAvidin. The amino acid sequence identity of rhizAvidin in relation to other known Avidin-like proteins is 20-30%. The amino acid residues involved in (strept)Avidin-biotin interaction are well conserved in rhizAvidin. The structural and functional properties of rhizAvidin were here carefully studied, rhizAvidin proving to share characteristics with both bradAvidin streptAvidin and Avidin. However, we found that it is the first naturally occurring dimeric protein in the Avidin protein family, in contrast to tetrameric (strept)Avidin and bradAvidin. Moreover, it possesses a proline after a flexible loop (GGSG) in a position close to Trp-110 in Avidin, which is an important biotin-binding residue. [ 3}H]biotin-dissociation and isothermal titration calorimetry (ITC) experiments showed dimeric rhizAvidin to be a high-affinity biotin-binding protein. Its thermal stability was lower than that of Avidin, though similar to streptAvidin it was insensitive to proteinase K. The immunological cross-reactivity of rhizAvidin was tested with human sera samples obtained from cancer patients exposed to (strept)Avidin. No significant cross-reactivity was observed. The biodistribution of the protein was studied by single photon emission computed tomography (SPECT) imaging in rats. Similar to Avidin, rhizAvidin was observed rapidly to accumulate mainly in the liver. Evidently, rhizAvidin could be used as a complement to (strept)Avidin in (strept)Avidin-biotin technology.
Olli H Laitinen - One of the best experts on this subject based on the ideXlab platform.
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Bacterial Avidins are a widely distributed protein family in Actinobacteria, Proteobacteria and Bacteroidetes
BMC Ecology and Evolution, 2021Co-Authors: Olli H Laitinen, Tanja P. Kuusela, Sampo Kukkurainen, Anssi Nurminen, Aki Sinkkonen, Vesa P HytonenAbstract:Background Avidins are biotin-binding proteins commonly found in the vertebrate eggs. In addition to streptAvidin from Streptomyces Avidinii , a growing number of Avidins have been characterized from divergent bacterial species. However, a systematic research concerning their taxonomy and ecological role has never been done. We performed a search for Avidin encoding genes among bacteria using available databases and classified potential Avidins according to taxonomy and the ecological niches utilized by host bacteria. Results Numerous Avidin-encoding genes were found in the phyla Actinobacteria and Proteobacteria. The diversity of protein sequences was high and several new variants of genes encoding biotin-binding Avidins were found. The living strategies of bacteria hosting Avidin encoding genes fall mainly into two categories. Human and animal pathogens were overrepresented among the found bacteria carrying Avidin genes. The other widespread category were bacteria that either fix nitrogen or live in root nodules/rhizospheres of plants hosting nitrogen-fixing bacteria. Conclusions Bacterial Avidins are a taxonomically and ecologically diverse group mainly found in Actinobacteria, Proteobacteria and Bacteroidetes, associated often with plant invasiveness. Avidin encoding genes in plasmids hint that Avidins may be horizontally transferred. The current survey may be used as a basis in attempts to understand the ecological significance of biotin-binding capacity.
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Brave new (strept)Avidins in biotechnology
Trends in biotechnology, 2007Co-Authors: Olli H Laitinen, Vesa P Hytonen, Henri R Nordlund, Markku S. KulomaaAbstract:Avidin and streptAvidin are widely used in (strept)Avidin–biotin technology, which is based on their tight biotin-binding capability. These techniques are exceptionally diverse, ranging from simple purification and labeling methods to sophisticated drug pre-targeting and nanostructure-building approaches. Improvements in protein engineering have provided new possibilities to develop tailored protein tools. The (strept)Avidin scaffold has been engineered to extend the existing range of applications and to develop new ones. Modifications to (strept)Avidins – such as simple amino acid substitutions to reduce biotin binding and alter physico–chemical characters – have recently developed into more sophisticated changes, including chimeric (strept)Avidins, topology rearrangements and stitching of non-natural amino acids into the active sites. In this review, we highlight the current status in genetically engineered (strept)Avidins and illustrate their versatility as advanced tools in the multiple fields of modern bioscience, medicine and nanotechnology.
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Genetically engineered Avidins and streptAvidins.
Cellular and molecular life sciences : CMLS, 2006Co-Authors: Olli H Laitinen, Vesa P Hytonen, Henri R Nordlund, Markku S. KulomaaAbstract:Chicken Avidin and bacterial streptAvidin, (strept)Avidin, are proteins widely utilized in a number of applications in life science, ranging from purification and labeling techniques to diagnostics, and from targeted drug delivery to nanotechnology. (Strept)Avidin-biotin technology relies on the extremely tight and specific affinity between (strept)Avidin and biotin (dissociation constant, Kd≈10−14–10−16 M). (Strept)Avidins are also exceptionally stable proteins. To study their ligand binding and stability characteristics, the two proteins have been extensively modified both chemically and genetically. There are excellent accounts of this technology and chemically modified (strept)Avidins, but no comprehensive reviews exist concerning genetically engineered (strept)Avidins. To fill this gap, we here go through the genetically engineered (strept)Avidins, summarizing how these constructs were designed and how they have improved our understanding of the structural and functional characteristics of these proteins, and the benefits they have provided for (strept)Avidin-biotin technology.
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Tetravalent single-chain Avidin: from subunits to protein domains via circularly permuted Avidins.
Biochemical Journal, 2005Co-Authors: Henri R Nordlund, Vesa P Hytonen, Olli H Laitinen, Juha A E Maatta, Jarno Hörhä, Eevaleena J. Porkka, Daniel J. White, Katrin K. Halling, J. Peter Slotte, Markku S. KulomaaAbstract:scAvd (single-chain Avidin, where two dcAvd are joined in a single polypeptide chain), having four biotin-binding domains, was constructed by fusion of topologically modified Avidin units. scAvd showed similar biotin binding and thermal stability properties as chicken Avidin. The DNA construct encoding scAvd contains four circularly permuted Avidin domains, plus short linkers connecting the four domains into a single polypeptide chain. In contrast with wild-type Avidin, which contains four identical Avidin monomers, scAvd enables each one of the four Avidin domains to be independently modified by protein engineering. Therefore the scAvd scaffold can be used to construct spatially and stoichiometrically defined pseudotetrameric Avidin molecules showing different domain characteristics. In addition, unmodified scAvd could be used as a fusion partner, since it provides a unique non-oligomeric structure, which is fully functional with four high-affinity biotin-binding sites. Furthermore, the subunit-to-domain strategy described in the present study could be applied to other proteins and protein complexes, facilitating the development of sophisticated protein tools for applications in nanotechnology and life sciences.
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Dual-affinity Avidin molecules.
Proteins, 2005Co-Authors: Vesa P Hytonen, Ari T Marttila, Henri R Nordlund, Thomas K M Nyholm, Jarno Hörhä, David E. Hyre, Tuomas Kulomaa, Eevaleena J. Porkka, Patrick S. Stayton, Olli H LaitinenAbstract:A recently reported dual-chain Avidin was modified further to contain two distinct, independent types of ligand-binding sites within a single polypeptide chain. Chicken Avidin is normally a tetrameric glycoprotein that binds water-soluble d-biotin with extreme affinity (Kd ≈ 10−15M). Avidin is utilized in various applications and techniques in the life sciences and in the nanosciences. In a recent study, we described a novel Avidin monomer-fusion chimera that joins two circularly permuted monomers into a single polypeptide chain. Two of these dual-chain Avidins were observed to associate spontaneously to form a dimer equivalent to the wt tetramer. In the present study, we successfully used this scaffold to generate Avidins in which the neighboring biotin-binding sites of dual-chain Avidin exhibit two different affinities for biotin. In these novel Avidins, one of the two binding sites in each polypeptide chain, the pseudodimer, is genetically modified to have lower binding affinity for biotin, whereas the remaining binding site still exhibits the high-affinity characteristic of the wt protein. The pseudotetramer (i.e., a dimer of dual-chain Avidins) has two high and two lower affinity biotin-binding sites. The usefulness of these novel proteins was demonstrated by immobilizing dual-affinity Avidin with its high-affinity sites. The sites with lower affinity were then used for affinity purification of a biotinylated enzyme. These “dual-affinity” Avidin molecules open up wholly new possibilities in Avidin–biotin technology, where they may have uses as novel bioseparation tools, carrier proteins, or nanoscale adapters. Proteins 2005. © 2005 Wiley-Liss, Inc.
