Strategies for the purification and characterization of protein scaffolds for the production of hybrid nanobiomaterials

Plascencia-Villa, Germán; Mena, Jimmy A.; Castro-Acosta, Ricardo M.; Fabián, Julio César; Ramı́rez, Octavio T.; Palomares, Laura A.

doi:10.1016/j.jchromb.2011.03.027

Cited by 23 publications

(22 citation statements)

References 26 publications

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“…Size exclusion chromatography (SEC) analysis showed two populations (Figure 1 B), one that migrated at the column exclusion limit (peak 1), which corresponded to VP6 nanotubes (VP6 NT ), and a second one with smaller size identified as VP6 U (peak 2), containing unassembled VP6 monomers and trimers. The population containing VP6 NT constituted 95% of the total protein, which is a typical value obtained with this purification process [ 3 , 29 ]. The presence of VP6 NT structures was confirmed by TEM (Figure 1 C).…”

Section: Resultsmentioning

confidence: 99%

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Effect of metal catalyzed oxidation in recombinant viral protein assemblies

et al. 2014

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BackgroundProtein assemblies, such as virus-like particles, have increasing importance as vaccines, delivery vehicles and nanomaterials. However, their use requires stable assemblies. An important cause of loss of stability in proteins is oxidation, which can occur during their production, purification and storage. Despite its importance, very few studies have investigated the effect of oxidation in protein assemblies and their structural units. In this work, we investigated the role of in vitro oxidation in the assembly and stability of rotavirus VP6, a polymorphic protein.ResultsThe susceptibility to oxidation of VP6 assembled into nanotubes (VP6NT) and unassembled VP6 (VP6U) was determined and compared to bovine serum albumin (BSA) as control. VP6 was more resistant to oxidation than BSA, as determined by measuring protein degradation and carbonyl content. It was found that assembly protected VP6 from in vitro metal-catalyzed oxidation. Oxidation provoked protein aggregation and VP6NT fragmentation, as evidenced by dynamic light scattering and transmission electron microscopy. Oxidative damage of VP6 correlated with a decrease of its center of fluorescence spectral mass. The in vitro assembly efficiency of VP6U into VP6NT decreased as the oxidant concentration increased.ConclusionsOxidation caused carbonylation, quenching, and destruction of aromatic amino acids and aggregation of VP6 in its assembled and unassembled forms. Such modifications affected protein functionality, including its ability to assemble. That assembly protected VP6 from oxidation shows that exposure of susceptible amino acids to the solvent increases their damage, and therefore the protein surface area that is exposed to the solvent is determinant of its susceptibility to oxidation. The inability of oxidized VP6 to assemble into nanotubes highlights the importance of avoiding this modification during the production of proteins that self-assemble. This is the first time that the role of oxidation in protein assembly is studied, evidencing that oxidation should be minimized during the production process if VP6 nanotubes are required.

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Section: Resultsmentioning

confidence: 99%

“…To further understand the effect of oxidation in VP6 NT , oxidized samples were analyzed by SEC, as previously described [ 29 , 33 ]. Absorbance at 280 nm and fluorescence of aromatic amino acids were followed (Figure 6 ).…”

Section: Resultsmentioning

confidence: 99%

Effect of metal catalyzed oxidation in recombinant viral protein assemblies

et al. 2014

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“…Therefore, our previous data on viral capsids can serve as a workflow to identify candidate proteins or enzymes for construction of BEI devices. Altogether, theoretical and experimental reports on BEI [41][42][43] are examples of how structural information could help to elucidate the behavior of biological systems during electrochemical reactions. Nonetheless, the aforementioned studies depend on structural information from biomolecules, and certain biomolecules are very complex and highly labile, which difficult elucidation of atomic positions.…”

Section: Molecular Dynamics As a Tool To Predict The Electrochemical mentioning

confidence: 99%

Design of Bioelectrochemical Interfaces Assisted by Molecular Dynamics Simulations

Vidal-Limón¹,

Huerta-Miranda²,

García-García³

et al. 2021

Homology Molecular Modeling - Perspectives and Applications

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The design of bioelectrochemical interfaces (BEI) is an interesting topic that recently demands attention. The synergy between biomolecules and chemical components is necessary to achieve high molecular selectivity and sensitivity for the development of biosensors, synthesis of different compounds, or catalytic processes. For most BEI, the charge transfer process occurs in environments with particular chemical conditions; modeling these environments is a challenging task and requires multidisciplinary efforts. These interfaces can be composed of biomolecules, such as proteins, DNA, or more complex systems like microorganisms. Oxidoreductases enzymes are good candidates, among others, due to their catalytic activities and structural characteristics. In BEI, enzymes are immobilized on conductive surfaces to improve charge transfer processes. Covalent immobilization is the most common method to prolong lifetime or modulate the detection process. However, it is necessary to implement new methodologies that allow the selection of the best candidates for a more efficient design. Homology modeling of oxidoreductases combined with Molecular Dynamics (MD) simulation methods are alternative and already routinely used tools to investigate the structure, dynamics, and thermodynamics of biological molecules. Our motivation is to show different techniques of molecular modeling (Homology Modeling, Gaussian accelerated molecular dynamics, directed adaptive molecular dynamics and electrostatic surface calculations), and using horseradish peroxidase as a model to understand the interactions between biomolecules and gold nanoclusters (as current collector). Additionally, we present our previous studies considering molecular simulations and we discuss recent advances in biomolecular simulations aimed at biosensor design.

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“…Self-assembled nanotubes were purified by continuous steps of ultrafiltration (Amicon, 30000 NMWL, EMD Millipore, Darmstadt, Germany), ion exchange chromatography (Q-Sepharose, Amersham, GE Healthcare Bio-Sciences, Pittsburgh PA, USA), and size-exclusion chromatography (HW65F, TOSOH Bioscience, Grove City, OH, USA) [18].…”

Section: Recombinant Production Of Viral Templatesmentioning

confidence: 99%

“…Additionally, genetically engineered M13 bacteriophages were used as biotemplates for Pd particles obtaining hybrid nanorods and nanowires with potential applications in nanobatteries [16,17]. Rotavirus VP6 protein, which composes the middle layer of the viral capsid, has shown versatility as a biotemplate to produce noble metal nanoparticles, including palladium nanoparticles of less than 5 nm in diameter synthesized over the external side of the VP6 nanotubes [18,19], and also the formation of silver nanowires in the interior of the VP6 nanotubes [20]. VP6 assemblies possess an inner capacity for the binding of metal ions (Ca 2+ and Zn 2+ ), but the specific residues where these interactions take place for functional metals like palladium or platinum are mostly unknown.…”

Section: Introductionmentioning

confidence: 99%

Molecular Docking and Aberration-Corrected STEM of Palladium Nanoparticles on Viral Templates

Carreño-Fuentes¹,

Bahena

Palomares³

et al. 2016

Metals

Self Cite

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Viral templates are highly versatile biotemplates used for the synthesis of nanostructured materials. Rotavirus VP6 self-assembles into nanotubular hollow structures with well-defined diameters and variable lengths, serving as a nucleic acid-free biotemplate to synthesize metal nanoparticles of controlled size, shape, and orientation. Molecular docking simulations show that exposed residues (H173-S240-D242 and N200-N310) of VP6 have the ability to specifically bind Pd(II) ions, which serve as nucleation sites for the growth and stabilization of palladium nanoclusters. Using VP6 nanotubes as biotemplates allows for obtaining small Pd particles of 1-5 nm in diameter. Advanced electron microscopy imaging and characterization through ultra-high-resolution field-emission scanning electron microscopy (UHR-FE-SEM) and spherical aberration-corrected scanning transmission electron microscopy (Cs-STEM) at a low voltage dose (80 kV) reveals, with high spatial resolution, the structure of Pd nanoparticles attached to the macromolecular biotemplates.

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Strategies for the purification and characterization of protein scaffolds for the production of hybrid nanobiomaterials

Cited by 23 publications

References 26 publications

Effect of metal catalyzed oxidation in recombinant viral protein assemblies

Effect of metal catalyzed oxidation in recombinant viral protein assemblies

Design of Bioelectrochemical Interfaces Assisted by Molecular Dynamics Simulations

Molecular Docking and Aberration-Corrected STEM of Palladium Nanoparticles on Viral Templates

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