Structural Adaptation of a Thermostable Biotin-binding Protein in a Psychrophilic Environment

Meir, Amit; Bayer, Edward A.; Livnah, Oded

doi:10.1074/jbc.m112.357186

Cited by 22 publications

(72 citation statements)

References 48 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Prior to measurements, proteins were dialyzed overnight against a solution containing 10 mM NaH 2 PO 4 . CD melting curves were measured at ks of 233 nm (apo form) and 235 nm (biotin-complex form), where the maximum difference between the native and unfolded forms appeared to be, similar to that previously reported for shwanavidin (Meir et al, 2012). T m values were calculated using Origin 8.1 software by fitting the T m curves of all hoefavidin forms to the Boltzman model and calculating its midpoint as the T m .…”

Section: Affinity and Thermostability Measurementsmentioning

confidence: 88%

“…The affinities of intact and short hoefavidin toward 2-iminobiotin were measured by surface plasmon resonance (SPR) using a Biacore 3000 optical biosensor instrument (Biacore AB, Uppsala, Sweden) as previously described for shwanavidin (Meir et al, 2012). Binding of the peptides to short hoefavidin was studied using Isothermal Titration Calorimetry (ITC).…”

Section: Affinity and Thermostability Measurementsmentioning

confidence: 99%

“…Novel proteins were discovered, among them avidin-related proteins (AVRs) (Laitinen et al, 2002) and bradavidin (Helppolainen et al, 2008;Leppiniemi et al, 2012;Nordlund et al, 2005), all exhibiting a tetrameric assembly. More recent studies, however, have revealed additional biotin-binding proteins, which display dimeric rather than tetrameric arrangements, while maintaining their high-affinity binding of biotin (Helppolainen et al, 2007;Meir et al, 2012Meir et al, , 2009. In this context, dimeric rhizavidin and shwanavidin exhibit similar tertiary topologies as their tetrameric relatives (Helppolainen et al, 2007;Meir et al, 2012Meir et al, , 2009.…”

Section: Introductionmentioning

confidence: 99%

“…More recent studies, however, have revealed additional biotin-binding proteins, which display dimeric rather than tetrameric arrangements, while maintaining their high-affinity binding of biotin (Helppolainen et al, 2007;Meir et al, 2012Meir et al, , 2009. In this context, dimeric rhizavidin and shwanavidin exhibit similar tertiary topologies as their tetrameric relatives (Helppolainen et al, 2007;Meir et al, 2012Meir et al, , 2009. The quaternary structures of the dimeric avidins are stabilized by the 1-4 interaction and maintain the high affinity toward biotin, rendering the essential Trp residue of the 1-2 interaction in tetrameric avidin redundant (Meir et al, 2012(Meir et al, , 2009.…”

Section: Introductionmentioning

confidence: 99%

“…In this context, dimeric rhizavidin and shwanavidin exhibit similar tertiary topologies as their tetrameric relatives (Helppolainen et al, 2007;Meir et al, 2012Meir et al, , 2009. The quaternary structures of the dimeric avidins are stabilized by the 1-4 interaction and maintain the high affinity toward biotin, rendering the essential Trp residue of the 1-2 interaction in tetrameric avidin redundant (Meir et al, 2012(Meir et al, , 2009.…”

Section: Introductionmentioning

confidence: 99%

See 4 more Smart Citations

Hoefavidin: A dimeric bacterial avidin with a C-terminal binding tail

Avraham

Meir

Fish

et al. 2015

Journal of Structural Biology

Self Cite

View full text Add to dashboard Cite

Section: Affinity and Thermostability Measurementsmentioning

confidence: 88%

Section: Affinity and Thermostability Measurementsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 3 more Smart Citations

Hoefavidin: A dimeric bacterial avidin with a C-terminal binding tail

Avraham

Meir

Fish

et al. 2015

Journal of Structural Biology

Self Cite

View full text Add to dashboard Cite

Stable, high‐affinity streptavidin monomer for protein labeling and monovalent biotin detection

Lim

Huang

Pralle

et al. 2012

Biotech & Bioengineering

122

115

View full text Add to dashboard Cite

The coupling between the quaternary structure, stability and function of streptavidin makes it difficult to engineer a stable, high affinity monomer for biotechnology applications. For example, the binding pocket of streptavidin tetramer is comprised of residues from multiple subunits, which cannot be replicated in a single domain protein. However, rhizavidin from Rhizobium etli was recently shown to bind biotin with high affinity as a dimer without the hydrophobic tryptophan lid donated by an adjacent subunit. In particular, the binding site of rhizavidin uses residues from a single subunit to interact with bound biotin. We therefore postulated that replacing the binding site residues of streptavidin monomer with corresponding rhizavidin residues would lead to the design of a high affinity monomer useful for biotechnology applications. Here, we report the construction and characterization of a structural monomer, mSA, which combines the streptavidin and rhizavidin sequences to achieve optimized biophysical properties. First, the biotin affinity of mSA (Kd = 2.8 nM) is the highest among nontetrameric streptavidin, allowing sensitive monovalent detection of biotinylated ligands. The monomer also has significantly higher stability (Tm = 59.8°C) and solubility than all other previously engineered monomers to ensure the molecule remains folded and functional during its application. Using fluorescence correlation spectroscopy, we show that mSA binds biotinylated targets as a monomer. We also show that the molecule can be used as a genetic tag to introduce biotin binding capability to a heterologous protein. For example, recombinantly fusing the monomer to a cell surface receptor allows direct labeling and imaging of transfected cells using biotinylated fluorophores. A stable and functional streptavidin monomer, such as mSA, should be a useful reagent for designing novel detection systems based on monovalent biotin interaction. Biotechnol. Bioeng. 2013; 110: 57–67. © 2012 Wiley Periodicals, Inc.

show abstract

The highly dynamic oligomeric structure of bradavidin II is unique among avidin proteins

et al. 2013

Self Cite

View full text Add to dashboard Cite

Bradavidin II is a biotin-binding protein from Bradyrhizobium japonicum that resembles chicken avidin and bacterial streptavidin. A biophysical characterization was carried out using dynamic light scattering, native mass spectrometry, differential scanning calorimetry, and isothermal titration calorimetry combined with structural characterization using X-ray crystallography. These observations revealed that bradavidin II differs from canonical homotetrameric avidin protein family members in its quaternary structure. In contrast with the other avidins, bradavidin II appears to have a dynamic (transient) oligomeric state in solution. It is monomeric at low protein concentrations but forms higher oligomeric assemblies at higher concentrations. The crystal structure of bradavidin II revealed an important role for Phe42 in shielding the bound ligand from surrounding water molecules, thus functionally replacing the L7,8 loop essential for tight ligand binding in avidin and streptavidin. This bradavidin II characterization opens new avenues for oligomerizationindependent biotin-binding protein development.

show abstract

Structural Adaptation of a Thermostable Biotin-binding Protein in a Psychrophilic Environment

Cited by 22 publications

References 48 publications

Hoefavidin: A dimeric bacterial avidin with a C-terminal binding tail

Hoefavidin: A dimeric bacterial avidin with a C-terminal binding tail

Stable, high‐affinity streptavidin monomer for protein labeling and monovalent biotin detection

The highly dynamic oligomeric structure of bradavidin II is unique among avidin proteins

Contact Info

Product

Resources

About