Effects of low urea concentrations on protein-water interactions

Ferreira, Luísa A.; Povarova, Olga I.; Stepanenko, Olga V.; Sulatskaya, Anna I.; Madeira, Pedro P.; Kuznetsova, Irina M.; Turoverov, Konstantin K.; Uversky, Vladimir N.; Zaslavsky, Boris Y.

doi:10.1080/07391102.2015.1135823

Cited by 9 publications

(5 citation statements)

References 43 publications

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“…VNAR showed resistance not only to low temperature but also to 1 M urea (Figure 3f). In this study, 0.5 M urea exhibits protein denaturing effects [32]. It has also been reported that a high concentration of urea inhibits antigen-antibody reactions [33].…”

Section: Discussionsupporting

confidence: 59%

Novel Approach for Obtaining Variable Domain of New Antigen Receptor with Different Physicochemical Properties from Japanese Topeshark (Hemitriakis japanica)

Nakada-Masuta,

Takeda,

Uchida

2023

Marine Drugs

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Diverse candidate antibodies are needed to successfully identify therapeutic and diagnostic applications. The variable domain of IgNAR (VNAR), a shark single-domain antibody, has attracted attention owing to its favorable physicochemical properties. The phage display method used to screen for optimal VNARs loses sequence diversity because of the bias caused by the differential ease of protein expression in Escherichia coli. Here, we investigated a VNAR selection method that combined panning with various selection pressures and next-generation sequencing (NGS) analyses to obtain additional candidates. Drawing inspiration from the physiological conditions of sharks and the physicochemical properties of VNARs, we examined the effects of NaCl and urea concentrations, low temperature, and preheating at the binding step of panning. VNAR phage libraries generated from Japanese topeshark (Hemitriakis japanica) were enriched under these conditions. We then performed NGS analysis and attempted to select clones that were specifically enriched under each panning condition. The identified VNARs exhibited higher reactivity than those obtained by panning without selection pressure. Additionally, they possess physicochemical properties that reflect their respective selection pressures. These results can greatly enhance our understanding of VNAR properties and offer guidance for the screening of high-quality VNAR clones that are present at low frequencies.

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Section: Discussionsupporting

confidence: 59%

Novel Approach for Obtaining Variable Domain of New Antigen Receptor with Different Physicochemical Properties from Japanese Topeshark (Hemitriakis japanica)

Nakada-Masuta,

Takeda,

Uchida

2023

Marine Drugs

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“…We observed more complex dependence of sfGFP stability and refolding on the presence of crowding agents. In fact, our previous data of the analysis of the urea-induced unfolding of structurally-different globular proteins [33,34], and the results of this study allow the conclusion that even in the absence of direct interaction between a crowding agent and the target protein the excluded volume theory cannot always be used to explain the experimental data. In fact, we show here that all crowding agents used in this study do not affect the structure of sfGFP.…”

Section: Discussionmentioning

confidence: 76%

“…This hypothesis is supported by the observed increase in the degree of sfGFP aggregation during the protein unfolding-refolding processes with the increase in the molecular mass and concentration of PEGs. In fact, it is evident now that the behavior of biomolecules in the conditions of macromolecular crowding cannot be explained only within the frames of the excluded volume effects and/or direct interaction between tested biomolecules and crowding agents [28,29,33,34]. The changes in the sfGFP stability towards the GTC action and the capability of this protein to refold in the presence of different crowding agents are shown here to be dependent on the type and concentration of the polymer used as a crowding agent.…”

Section: Discussionmentioning

confidence: 99%

“…This further supports the idea that, in addition to the excluded volume, some additional factors are involved in the modulation of the behavior of sfGFP in the crowded milieu. Accumulated experimental data including results obtained in this study indicate that crowding agents may affect the solvent properties of aqueous solutions in different manners, depending on the crowding agent macromolecule structure and the concentration [33,34,55]. These solvent properties are the solvent dipolarity/polarizability, hydrogen-bond donor acidity, and hydrogen-bond acceptor basicity of aqueous solutions [55].…”

Section: Discussionmentioning

confidence: 99%

“…In laboratory practice, different polymer molecules, such as polyethylene glycol (PEG) of different molecular weights, Dextran, Ficoll, and inert proteins, are typically used as crowding agents [25,28,29,31]. We, and others, established that the excluded volume effect is not the only factor influencing the behavior of the protein in a crowded milieu [29,33,34]. Apparently, the special role belongs to the ability of the crowding agents to alter the properties of the solvent.…”

Section: Introductionmentioning

confidence: 99%

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Peculiarities of the Super-Folder GFP Folding in a Crowded Milieu

Stepanenko

Kuznetsova

Uversky

et al. 2016

IJMS

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The natural cellular milieu is crowded by large quantities of various biological macromolecules. This complex environment is characterized by a limited amount of unoccupied space, limited amounts of free water, and changed solvent properties. Obviously, such a tightly packed cellular environment is poorly mimicked by traditional physiological conditions, where low concentrations of a protein of interest are analyzed in slightly salted aqueous solutions. An alternative is given by the use of a model crowded milieu, where a protein of interest is immersed in a solution containing high concentrations of various polymers that serve as model crowding agents. An expected outcome of the presence of such macromolecular crowding agents is their ability to increase conformational stability of a globular protein due to the excluded volume effects. In line with this hypothesis, the behavior of a query protein should be affected by the hydrodynamic size and concentration of an inert crowder (i.e., an agent that does not interact with the protein), whereas the chemical nature of a macromolecular crowder should not play a role in its ability to modulate conformational properties. In this study, the effects of different crowding agents (polyethylene glycols (PEGs) of various molecular masses (PEG-600, PEG-8000, and PEG-12000), Dextran-70, and Ficoll-70) on the spectral properties and unfolding–refolding processes of the super-folder green fluorescent protein (sfGFP) were investigated. sfGFP is differently affected by different crowders, suggesting that, in addition to the expected excluded volume effects, there are some changes in the solvent properties.

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