scholarly journals Generating a High Valency Biotin Binder by Selecting Uniform Protein Assemblies via Crystallization

Crystals ◽  
2019 ◽  
Vol 9 (7) ◽  
pp. 353
Author(s):  
Orly Avraham ◽  
Yael Levi-Kalisman ◽  
Oded Livnah
Keyword(s):  
Quaternary Structure ◽  
Disulfide Bridge ◽  
Symmetry Operation ◽  
Added Value ◽  
Purification Process ◽  
Protein Assemblies ◽  
High Affinity ◽  
Covalent Crosslinking ◽  
Crystallographic Symmetry ◽  
Biotin Binding

Crystallization is a common practice in the purification process in small molecule synthesis while selecting the wanted product. For proteins it is rarely applied due to the methodological predicaments in obtaining crystals. Our observation of the stabilized octamers in the crystal structure of hoefavidin, a novel dimeric member of the avidin family, led to the notion of developing a novel biotechnological tool via covalent crosslinking. The avidin–biotin system has been exploited for decades utilizing the ultra-high affinity between avidin and biotin as a basis for numerous applications. Optimizing the system led to the discovery of a novel group of dimeric avidins including hoefavidin. Hoefavidin has a dynamic quaternary structure, where a dimer is the basis for generating the octamer via crystallographic symmetry operation. Upon biotin binding in solution hoefavidin dissociates solely into dimers. In order to stabilize the octamer, we designed the P61C mutant to form a disulfide bridge stabilizing the octamer and preventing dissociation upon biotin binding. The process of selecting P61C hoefavidin uniform octamers includes crystallization followed by dissolving the crystals. The P61C modified hoefavidin octamer can have a substantial added value to the various biotechnological applications and advances of the biotin based high affinity systems.

scholarly journals Structural analysis of cross α-helical nanotubes provides insight into the designability of filamentous peptide nanomaterials

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Fengbin Wang ◽  
Ordy Gnewou ◽  
Charles Modlin ◽  
Leticia C. Beltran ◽  
Chunfu Xu ◽  
...  
Keyword(s):  
Structural Analysis ◽  
Synthetic Peptide ◽  
Quaternary Structure ◽  
De Novo ◽  
Rational Approach ◽  
Lateral Interactions ◽  
Protein Assemblies ◽  
Self Assembling ◽  
Arginine Residues ◽  
Insight Into

AbstractThe exquisite structure-function correlations observed in filamentous protein assemblies provide a paradigm for the design of synthetic peptide-based nanomaterials. However, the plasticity of quaternary structure in sequence-space and the lability of helical symmetry present significant challenges to the de novo design and structural analysis of such filaments. Here, we describe a rational approach to design self-assembling peptide nanotubes based on controlling lateral interactions between protofilaments having an unusual cross-α supramolecular architecture. Near-atomic resolution cryo-EM structural analysis of seven designed nanotubes provides insight into the designability of interfaces within these synthetic peptide assemblies and identifies a non-native structural interaction based on a pair of arginine residues. This arginine clasp motif can robustly mediate cohesive interactions between protofilaments within the cross-α nanotubes. The structure of the resultant assemblies can be controlled through the sequence and length of the peptide subunits, which generates synthetic peptide filaments of similar dimensions to flagella and pili.

scholarly journals Metal‐Templated Design of Chemically Switchable Protein Assemblies with High‐Affinity Coordination Sites

2020 ◽  
Vol 132 (49) ◽  
pp. 22124-22128
Author(s):  
Albert Kakkis ◽  
Derek Gagnon ◽  
Julian Esselborn ◽  
R. David Britt ◽  
F. Akif Tezcan
Keyword(s):  
Protein Assemblies ◽  
High Affinity ◽  
Coordination Sites

Structural origins of high-affinity biotin binding to streptavidin

Science ◽  
1989 ◽  
Vol 243 (4887) ◽  
pp. 85-88 ◽  
Author(s):  
P. Weber ◽  
D. Ohlendorf ◽  
J. Wendoloski ◽  
F. Salemme
Keyword(s):  
High Affinity ◽  
Biotin Binding

scholarly journals Metal‐Templated Design of Chemically Switchable Protein Assemblies with High‐Affinity Coordination Sites

2020 ◽  
Vol 59 (49) ◽  
pp. 21940-21944
Author(s):  
Albert Kakkis ◽  
Derek Gagnon ◽  
Julian Esselborn ◽  
R. David Britt ◽  
F. Akif Tezcan
Keyword(s):  
Protein Assemblies ◽  
High Affinity ◽  
Coordination Sites

scholarly journals Subunit composition of an energy-coupling-factor-type biotin transporter analysed in living bacteria

2010 ◽  
Vol 431 (3) ◽  
pp. 373-381 ◽  
Author(s):  
Friedrich Finkenwirth ◽  
Olivia Neubauer ◽  
Julia Gunzenhäuser ◽  
Janna Schoknecht ◽  
Silvia Scolari ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Quaternary Structure ◽  
Transmembrane Protein ◽  
Yellow Fluorescent Protein ◽  
Energy Coupling ◽  
Coupling Factor ◽  
Supramolecular Organization ◽  
High Affinity ◽  
Donor Acceptor ◽  
Living Bacteria

BioMNY, a bacterial high-affinity biotin transporter, is a member of the recently defined class of ECF (energy-coupling factor) transporters. These systems are composed of ABC (ATP-binding-cassette) ATPases (represented by BioM in the case of the biotin transporter), a universally conserved transmembrane protein (BioN) and a core transporter component (BioY), in unknown stoichiometry. The quaternary structure of BioY, which functions as a low-affinity biotin transporter in the absence of BioMN, and of BioMNY was investigated by a FRET (Förster resonance energy transfer) approach using living recombinant Escherichia coli cells. To this end, the donor–acceptor pair, of Cerulean and yellow fluorescent protein respectively, were fused to BioM, BioN and BioY. The fusion proteins were stable and the protein tags did not interfere with transport and ATPase activities. Specific donor–acceptor interactions were characterized by lifetime-based FRET spectroscopy. The results suggest an oligomeric structure for the solitary BioY core transporter and oligomeric forms of BioM and BioY in BioMNY complexes. We surmise that oligomers of BioY are the functional units of the low- and high-affinity biotin transporter in the living cell. Beyond its relevance for clarifying the supramolecular organization of ECF transporters, the results demonstrate the general applicability of lifetime-based FRET studies in living bacteria.

scholarly journals Mutation of the important Tyr-33 residue of chicken avidin: functional and structural consequences

2003 ◽  
Vol 369 (2) ◽  
pp. 249-254 ◽  
Author(s):  
Ari T. MARTTILA ◽  
Vesa P. HYTÖNEN ◽  
Olli H. LAITINEN ◽  
Edward A. BAYER ◽  
Meir WILCHEK ◽  
...  
Keyword(s):  
Ligand Binding ◽  
Quaternary Structure ◽  
Affinity Purification ◽  
Ph Dependence ◽  
Tight Binding ◽  
Carbonyl Oxygen ◽  
Binding Characteristics ◽  
Biotin Binding ◽  
High Ligand

The strong interaction between avidin and biotin is so tight (dissociation constant 10-15M) that conditions usually sufficient for protein denaturing fail to dislodge biotin from the avidin—biotin complex. This kind of irreversible binding hinders the use of avidin in applications such as affinity purification or protein immobilization. To address this concern, we have constructed a series of mutants of the strategically positioned Tyr-33 in order to study the role of this residue in biotin binding, and to create avidin variants with more reversible ligand-binding properties. Unexpectedly, an avidin mutant in which Tyr-33 was replaced with phenylalanine (Avm-Y33F) displayed similar biotin-binding characteristics to the native avidin, indicating that the hydrogen bond formed between the hydroxy group of Tyr-33 and the carbonyl oxygen of biotin is not as important for the tight binding of biotin as previously suggested. In terms of the reversibility of biotin binding, Avm-Y33H was the most successful substitution constructed in this study. Interestingly, the binding of this mutant exhibited clear pH-dependence, since at neutral pH it bound to the biotin surface in an irreversible fashion, whereas, at pH9, 50% of the bound protein could be released with free biotin. Furthermore, although Tyr-33 is located relatively distant from the monomer—monomer interfaces, the mutagenesis of this residue also weakened the quaternary structure of avidin, indicating that the high ligand binding and the high stability of avidin have evolved together and it is difficult to modify one without affecting the other.

Structure of γ-conglutin: insight into the quaternary structure of 7S basic globulins from legumes

2015 ◽  
Vol 71 (2) ◽  
pp. 224-238 ◽  
Author(s):  
Jaroslaw Czubinski ◽  
Jakub Barciszewski ◽  
Miroslaw Gilski ◽  
Kamil Szpotkowski ◽  
Janusz Debski ◽  
...  
Keyword(s):  
Plant Cell Wall ◽  
Quaternary Structure ◽  
Physiological Role ◽  
Structural Similarity ◽  
Disulfide Bridge ◽  
Structural Basis ◽  
Evolutionary Context ◽  
Lupin Seeds ◽  
High Level ◽  
Quaternary Assembly

γ-Conglutin from lupin seeds is an unusual 7S basic globulin protein. It is capable of reducing glycaemia in mammals, but the structural basis of this activity is not known. γ-Conglutin shares a high level of structural homology with glycoside hydrolase inhibitor proteins, although it lacks any kind of inhibitory activity against plant cell-wall degradation enzymes. In addition, γ-conglutin displays a less pronounced structural similarity to pepsin-like aspartic proteases, but it is proteolytically dysfunctional. Only one structural study of a legume 7S basic globulin, that isolated from soybean, has been reported to date. The quaternary assembly of soybean 7S basic globulin (Bg7S) is arranged as a cruciform-shaped tetramer comprised of two superposed dimers. Here, the crystal structure of γ-conglutin isolated fromLupinus angustifoliusseeds (LangC) is presented. The polypeptide chain of LangC is post-translationally cleaved into α and β subunits but retains its covalent integrity owing to a disulfide bridge. The protomers of LangC undergo an intricate quaternary assembly, resulting in a ring-like hexamer with noncrystallographicD3symmetry. The twofold-related dimers are similar to those in Bg7S but their assembly is different as a consequence of mutations in a β-strand that is involved in intermolecular β-sheet formation in γ-conglutin. Structural elucidation of γ-conglutin will help to explain its physiological role, especially in the evolutionary context, and will guide further research into the hypoglycaemic activity of this protein in humans, with potential consequences for novel antidiabetic therapies.

scholarly journals Introduction of histidine residues into avidin subunit interfaces allows pH-dependent regulation of quaternary structure and biotin binding

FEBS Letters ◽  
2003 ◽  
Vol 555 (3) ◽  
pp. 449-454 ◽  
Author(s):  
Henri R Nordlund ◽  
Vesa P Hytönen ◽  
Olli H Laitinen ◽  
Sanna T.H Uotila ◽  
Einari A Niskanen ◽  
...  
Keyword(s):  
Quaternary Structure ◽  
Histidine Residues ◽  
Biotin Binding ◽  
Ph Dependent
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