scholarly journals Structure and stability of recombinant bovine odorant-binding protein: I. Design and analysis of monomeric mutants

PeerJ ◽  
2016 ◽  
Vol 4 ◽  
pp. e1933 ◽  
Author(s):  
Olga V. Stepanenko ◽  
Denis O. Roginskii ◽  
Olesya V. Stepanenko ◽  
Irina M. Kuznetsova ◽  
Vladimir N. Uversky ◽  
...  
Keyword(s):  
Disulfide Bond ◽  
Binding Protein ◽  
Stable State ◽  
Structural Features ◽  
Odorant Binding Protein ◽  
Domain Swapping ◽  
Tryptophan Residues ◽  
Monomeric Structure ◽  
Odorant Binding

Bovine odorant-binding protein (bOBP) differs from other lipocalins by lacking the conserved disulfide bond and for being able to form the domain-swapped dimers. To identify structural features responsible for the formation of the bOBP unique dimeric structure and to understand the role of the domain swapping on maintaining the native structure of the protein, structural properties of the recombinant wild type bOBP and its mutant that cannot dimerize via the domain swapping were analyzed. We also looked at the effect of the disulfide bond by designing a monomeric bOBPs with restored disulfide bond which is conserved in other lipocalins. Finally, to understand which features in the microenvironment of the bOBP tryptophan residues play a role in the defining peculiarities of the intrinsic fluorescence of this protein we designed and investigated single-tryptophan mutants of the monomeric bOBP. Our analysis revealed that the insertion of the glycine after the residue 121 of the bOBP prevents domain swapping and generates a stable monomeric protein bOBP-Gly121+. We also show that the restored disulfide bond in the GCC-bOBP mutant leads to the noticeable stabilization of the monomeric structure. Structural and functional analysis revealed that none of the amino acid substitutions introduced to the bOBP affected functional activity of the protein and that the ligand binding leads to the formation of a more compact and stable state of the recombinant bOBP and its mutant monomeric forms. Finally, analysis of the single-tryptophan mutants of the monomeric bOBP gave us a unique possibility to find peculiarities of the microenvironment of tryptophan residues which were not previously described.

scholarly journals Structure and stability of recombinant bovine odorant-binding protein: I. Design and analysis of monomeric mutants

2015 ◽  
Author(s):  
Olga V Stepanenko ◽  
Denis O Roginskii ◽  
Olesya V Stepanenko ◽  
Irina M Kuznetsova ◽  
Vladimir N Uversky ◽  
...  
Keyword(s):  
Disulfide Bond ◽  
Binding Protein ◽  
Stable State ◽  
Structural Features ◽  
Odorant Binding Protein ◽  
Domain Swapping ◽  
Tryptophan Residues ◽  
Monomeric Structure ◽  
Odorant Binding

Bovine odorant-binding protein (bOBP) differs from other lipocalins by lacking the conserved disulfide bond and being able to form the domain-swapped dimers. To identify structural features responsible for the formation of the bOBP unique dimeric structure and to understand the role of the domain swapping on maintaining the native structure of the protein, structural properties of the recombinant wild type bOBP and its mutant that cannot dimerize via the domain swapping were analyzed. We also looked at the effect of the disulfide bond by designing a monomeric bOBPs with restored disulfide bond which is conserved in other lipocalins. Finally, to understand which features in the microenvironment of the bOBP tryptophan residues play a role in the defining peculiarities of the intrinsic fluorescence of this protein we designed and investigated single-tryptophan mutants of the monomeric bOBP. Our analysis revealed that the insertion of the glycine after the residue 121 of the bOBP prevents domain swapping and generates a stable monomeric protein bOBP-Gly121+. We also show that the restored disulfide bond in the GCC-bOBP mutant leads to the noticeable stabilization of the monomeric structure. Structural and functional analysis revealed that none of the amino acid substitutions introduced to the bOBP affected functional activity of the protein and that the ligand binding leads to the formation of a more compact and stable state of the recombinant bOBP and its mutant monomeric forms. Finally, analysis of the single-tryptophan mutants of the monomeric bOBP gave us a unique possibility to find peculiarities of the microenvironment of tryptophan residues which were not previously described.

scholarly journals Structure and stability of recombinant bovine odorant-binding protein: I. Design and analysis of monomeric mutants

2015 ◽  
Author(s):  
Olga V Stepanenko ◽  
Denis O Roginskii ◽  
Olesya V Stepanenko ◽  
Irina M Kuznetsova ◽  
Vladimir N Uversky ◽  
...  
Keyword(s):  
Disulfide Bond ◽  
Binding Protein ◽  
Stable State ◽  
Structural Features ◽  
Odorant Binding Protein ◽  
Domain Swapping ◽  
Tryptophan Residues ◽  
Monomeric Structure ◽  
Odorant Binding

Bovine odorant-binding protein (bOBP) differs from other lipocalins by lacking the conserved disulfide bond and being able to form the domain-swapped dimers. To identify structural features responsible for the formation of the bOBP unique dimeric structure and to understand the role of the domain swapping on maintaining the native structure of the protein, structural properties of the recombinant wild type bOBP and its mutant that cannot dimerize via the domain swapping were analyzed. We also looked at the effect of the disulfide bond by designing a monomeric bOBPs with restored disulfide bond which is conserved in other lipocalins. Finally, to understand which features in the microenvironment of the bOBP tryptophan residues play a role in the defining peculiarities of the intrinsic fluorescence of this protein we designed and investigated single-tryptophan mutants of the monomeric bOBP. Our analysis revealed that the insertion of the glycine after the residue 121 of the bOBP prevents domain swapping and generates a stable monomeric protein bOBP-Gly121+. We also show that the restored disulfide bond in the GCC-bOBP mutant leads to the noticeable stabilization of the monomeric structure. Structural and functional analysis revealed that none of the amino acid substitutions introduced to the bOBP affected functional activity of the protein and that the ligand binding leads to the formation of a more compact and stable state of the recombinant bOBP and its mutant monomeric forms. Finally, analysis of the single-tryptophan mutants of the monomeric bOBP gave us a unique possibility to find peculiarities of the microenvironment of tryptophan residues which were not previously described.

Molecular and Functional Characterization of an Odorant Binding Protein of the Asian Elephant,Elephas maximus: Implications for the Role of Lipocalins in Mammalian Olfaction†

Biochemistry ◽  
2002 ◽  
Vol 41 (39) ◽  
pp. 11786-11794 ◽  
Author(s):  
Josef Lazar ◽  
David R. Greenwood ◽  
L. E. L. Rasmussen ◽  
Glenn D. Prestwich
Keyword(s):  
Binding Protein ◽  
Functional Characterization ◽  
Odorant Binding Protein ◽  
Asian Elephant ◽  
Elephas Maximus ◽  
Odorant Binding

Domain swapping creates a third putative combining site in bovine odorant binding protein dimer

1996 ◽  
Vol 3 (10) ◽  
pp. 863-867 ◽  
Author(s):  
Mariella Tegoni ◽  
Roberto Ramoni ◽  
Enrico Bignetti ◽  
Silvia Spinelli ◽  
Christian Cambillau
Keyword(s):  
Binding Protein ◽  
Odorant Binding Protein ◽  
Domain Swapping ◽  
Protein Dimer ◽  
Odorant Binding

The Structure of the Monomeric Porcine Odorant Binding Protein Sheds Light on the Domain Swapping Mechanism‡

Biochemistry ◽  
1998 ◽  
Vol 37 (22) ◽  
pp. 7913-7918 ◽  
Author(s):  
Silvia Spinelli ◽  
Roberto Ramoni ◽  
Stefano Grolli ◽  
Jacques Bonicel ◽  
Christian Cambillau ◽  
...  
Keyword(s):  
Binding Protein ◽  
Odorant Binding Protein ◽  
Domain Swapping ◽  
Odorant Binding ◽  
Porcine Odorant Binding Protein

scholarly journals Expression in Antennae and Reproductive Organs Suggests a Dual Role of an Odorant-Binding Protein in Two Sibling Helicoverpa Species

PLoS ONE ◽  
2012 ◽  
Vol 7 (1) ◽  
pp. e30040 ◽  
Author(s):  
Ya-Lan Sun ◽  
Ling-Qiao Huang ◽  
Paolo Pelosi ◽  
Chen-Zhu Wang
Keyword(s):  
Binding Protein ◽  
Reproductive Organs ◽  
Dual Role ◽  
Odorant Binding Protein ◽  
Odorant Binding

scholarly journals Structure and stability of recombinant bovine odorant-binding protein: III. Peculiarities of the wild type bOBP unfolding in crowded milieu

PeerJ ◽  
2016 ◽  
Vol 4 ◽  
pp. e1642 ◽  
Author(s):  
Olga V. Stepanenko ◽  
Denis O. Roginskii ◽  
Olesya V. Stepanenko ◽  
Irina M. Kuznetsova ◽  
Vladimir N. Uversky ◽  
...  
Keyword(s):  
Disulfide Bond ◽  
Binding Protein ◽  
Guanidine Hydrochloride ◽  
Conformational Stability ◽  
Odorant Binding Protein ◽  
Stable Domain ◽  
Peg 4000 ◽  
Similar Chemical ◽  
High Concentrations ◽  
Odorant Binding

Contrary to the majority of the members of the lipocalin family, which are stable monomers with the specific OBP fold (a β-barrel consisting of a 8-stranded anti-parallel β-sheet followed by a short α-helical segment, a ninth β-strand, and a disordered C-terminal tail) and a conserved disulfide bond, bovine odorant-binding protein (bOBP) does not have such a disulfide bond and forms a domain-swapped dimer that involves crossing the α-helical region from each monomer over the β-barrel of the other monomer. Furthermore, although natural bOBP isolated from bovine tissues exists as a stable domain-swapped dimer, recombinant bOBP has decreased dimerization potential and therefore exists as a mixture of monomeric and dimeric variants. In this article, we investigated the effect model crowding agents of similar chemical nature but different molecular mass on conformational stability of the recombinant bOBP. These experiments were conducted in order to shed light on the potential influence of model crowded environment on the unfolding-refolding equilibrium. To this end, we looked at the influence of PEG-600, PEG-4000, and PEG-12000 in concentrations of 80, 150, and 300 mg/mL on the equilibrium unfolding and refolding transitions induced in the recombinant bOBP by guanidine hydrochloride. We are showing here that the effect of crowding agents on the structure and conformational stability of the recombinant bOBP depends on the size of the crowder, with the smaller crowding agents being more effective in the stabilization of the bOBP native dimeric state against the guanidine hydrochloride denaturing action. This effect of the crowding agents is concentration dependent, with the high concentrations of the agents being more effective.

scholarly journals Structure and stability of recombinant bovine odorant-binding protein: III. Peculiarities of the wild type bOBP unfolding in crowded milieu

2015 ◽  
Author(s):  
Olga V Stepanenko ◽  
Denis O Roginskii ◽  
Olesya V Stepanenko ◽  
Irina M Kuznetsova ◽  
Vladimir N Uversky ◽  
...  
Keyword(s):  
Disulfide Bond ◽  
Binding Protein ◽  
Guanidine Hydrochloride ◽  
Conformational Stability ◽  
Odorant Binding Protein ◽  
Stable Domain ◽  
Helical Segment ◽  
Peg 4000 ◽  
Similar Chemical ◽  
Odorant Binding

Contrarily to the majority of the members of the lipocalin family, which are stable monomers with the specific OBP fold (a β-barrel consisting of a 8-stranded anti-parallel β-sheet followed by a short α-helical segment, a ninth β-strand, and a disordered C-terminal tail) and a conserved disulfide bond, bovine odorant-binding protein (bOBP) does not have such a disulfide bond and forms a domain-swapped dimer that involves crossing the α-helical region from each monomer over the β-barrel of the other monomer. Furthermore, although natural bOBP isolated from bovine tissues exists as a stable domain-swapped dimer, recombinant bOBP has decreased dimerization potential and therefore exists as a mixture of monomeric and dimeric variants. In this article, we investigated the effect model crowding agents of similar chemical nature but different molecular mass on conformational stability of the recombinant bOBP. These experiments were conducted in order shed light on the potential influence of model crowded environment on the unfolding-refolding equilibrium. To this end, we looked at the influence of PEG-600, PEG-4000, and PEG-12000 in concentrations of 80, 150, and 300 mg/mL on the equilibrium unfolding and refolding transitions induced in the recombinant bOBP by guanidine hydrochloride.

scholarly journals Structure and stability of recombinant bovine odorant-binding protein: III. Peculiarities of the wild type bOBP unfolding in crowded milieu

2015 ◽  
Author(s):  
Olga V Stepanenko ◽  
Denis O Roginskii ◽  
Olesya V Stepanenko ◽  
Irina M Kuznetsova ◽  
Vladimir N Uversky ◽  
...  
Keyword(s):  
Disulfide Bond ◽  
Binding Protein ◽  
Guanidine Hydrochloride ◽  
Conformational Stability ◽  
Odorant Binding Protein ◽  
Stable Domain ◽  
Helical Segment ◽  
Peg 4000 ◽  
Similar Chemical ◽  
Odorant Binding

Contrarily to the majority of the members of the lipocalin family, which are stable monomers with the specific OBP fold (a β-barrel consisting of a 8-stranded anti-parallel β-sheet followed by a short α-helical segment, a ninth β-strand, and a disordered C-terminal tail) and a conserved disulfide bond, bovine odorant-binding protein (bOBP) does not have such a disulfide bond and forms a domain-swapped dimer that involves crossing the α-helical region from each monomer over the β-barrel of the other monomer. Furthermore, although natural bOBP isolated from bovine tissues exists as a stable domain-swapped dimer, recombinant bOBP has decreased dimerization potential and therefore exists as a mixture of monomeric and dimeric variants. In this article, we investigated the effect model crowding agents of similar chemical nature but different molecular mass on conformational stability of the recombinant bOBP. These experiments were conducted in order shed light on the potential influence of model crowded environment on the unfolding-refolding equilibrium. To this end, we looked at the influence of PEG-600, PEG-4000, and PEG-12000 in concentrations of 80, 150, and 300 mg/mL on the equilibrium unfolding and refolding transitions induced in the recombinant bOBP by guanidine hydrochloride.

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