A new additive for protein crystallization

Vuillard, Laurent; Rabilloud, Thierry; Leberman, Reuben; Berthet-Colominas, C.; Cusack, Stephen

doi:10.1016/0014-5793(94)01060-9

Cited by 31 publications

(23 citation statements)

References 15 publications

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“…The same trend was observed when the nanogel concentration was increased (Figure S5). In the presence of NG-F, less than 10% fibrillation is observed even after heating at high temperatures for 30 minutes, a value that is much lower than that obtained with linear polymers (poly-SPB) and previously reported compounds such as non-detergent sulfobetaines3334353637 and L-arginine hydrochloride3839 (Figure S6). …”

Section: Resultscontrasting

confidence: 54%

Inhibition of protein aggregation by zwitterionic polymer-based core-shell nanogels

Rajan

Matsumura

2017

Sci Rep

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Protein aggregation is a process by which misfolded proteins polymerizes into aggregates and forms fibrous structures with a β-sheet conformation, known as amyloids. It is an undesired outcome, as it not only causes numerous neurodegenerative diseases, but is also a major deterrent in the development of protein biopharmaceuticals. Here, we report a rational design for the synthesis of novel zwitterionic polymer-based core-shell nanogels via controlled radical polymerization. Nanogels with different sizes and functionalities in the core and shell were prepared. The nanogels exhibit remarkable efficiency in the protection of lysozyme against aggregation. Addition of nanogels suppresses the formation of toxic fibrils and also enables lysozyme to retain its enzymatic activity. Increasing the molecular weight and degree of hydrophobicity markedly increases its overall efficiency. Investigation of higher order structures revealed that lysozyme when heated without any additive loses its secondary structure and transforms into a random coil conformation. In contrast, presence of nanogels facilitates the retention of higher order structures by acting as molecular chaperones, thereby reducing molecular collisions. The present study is the first to show that it is possible to design zwitterionic nanogels using appropriate polymerization techniques that will protect proteins under conditions of extreme stress and inhibit aggregation.

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Section: Resultscontrasting

confidence: 54%

Inhibition of protein aggregation by zwitterionic polymer-based core-shell nanogels

Rajan

Matsumura

2017

Sci Rep

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“…We showed in previous works that the use of NDSB could be beneficial for the extraction of nuclear and membrane bound proteins [8,9] for the stabilization of halophilic proteins [10] and in protein crystallization [7,21]. NDSBs possess several properties that makes them particularly suited for protein work: they are zwitterionic, they are highly soluble in water, they do not alter significantly the pH or viscosity of biological buffers, they can easily be removed by dialysis since they do not form micelles, some of them even allow monitoring of protein concentration by measurement of absorbance at 280 nm.…”

Section: Discussionmentioning

confidence: 97%

“…The effects of NDSBs on folding can be related to what was observed in protein crystal growth in the presence of NDSB [7,21]. In the crystallisation process, NDSBs act by preventing the weak non-specific interchain interactions that lead to amorphous precipitation but usually fail to perturb the stronger specific interactions responsible for crystal growth.…”

Section: Discussionmentioning

confidence: 99%

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Interactions of non‐detergent sulfobetaines with early folding intermediates facilitate in vitro protein renaturation

Vuillard

Rabilloud

Goldberg

1998

European Journal of Biochemistry

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Non-detergent sulfobetaines (NDSB) are a family of solubilizing and stabilizing agents for proteins. In a previous study [Goldberg, M. E., Expert-Bezancon, N., Vuillard, L. & Rabilloud, T. (1996) Folding & Design 1, 21Ϫ27] we showed that the amount of active protein recovered in in vitro folding experiments could be significantly increased by some NDSBS. In this work we investigated the mechanisms by which these molecules facilitate protein renaturation. Stopped-flow and manual-mixing fluorescence and enzyme activity measurements were used to compare the kinetics of protein folding in the presence and absence of . Hen lysozyme and the β2 subunit of Escherichia coli tryptophan synthase were chosen as model systems since their folding pathways had been previously investigated in detail. It is shown that, massive aggregation of tryptophan synthase occurs within less than 2.5 s after dilution in the renaturation buffer, but can be prevented by NDSB 256 ; only very early folding phases (such as the formation of a loosely packed hydrophobic core able to bind 8-anilino-1-naphthalenesulphonic acid, and the initial burying of tryptophan 177) are significantly altered by NDSB256 ; none of the later phases is affected. Furthermore, NDSB 256 did not significantly affect any of the kinetic phases observed during the refolding of denatured lysozyme retaining intact disulphide bonds. This shows that NDSB 256 only interferes with very early steps in the folding process and acts by limiting the abortive interactions that could lead to the formation of inactive aggregates.Keywords : sulfobetaine; protein folding; lysozyme; tryptophan synthase.Understanding the molecular mechanisms of protein folding is one of the most challenging problems of modern molecular and cell biology. Besides its academic interest it is also a major issue in biotechnology, since overexpressed proteins are often obtained as insoluble aggregates that must be submitted to complex procedures aimed at generating their biologically active conformation. These procedures, as well as basic investigations on the folding mechanisms, are frequently severely hampered by the tendency of the denatured protein to aggregate when transferred into the renaturation buffer. It has been proposed [1Ϫ3] that during their folding, proteins undergo a kinetic partitioning between two alternative pathways, one leading to the native conformation, the other to aggregates. In this model, it is also assumed that the energy barrier between these two final states is high enough to render their interconversion practically impossible. Based on this conclusion, a variety of approaches has been used to favor renaturation over aggregation [4]. Most methods aim at minimizing aggregation, rather than at helping productive folding. For instance, one can, lower the protein concentration to reduce the rate of multimolecular reactions involved in aggreCorrespondence to M. E. Goldberg, Unité de Biochimie Cellulaire (CNRS URA 1129), Institut Pasteur, 28 rue du Dr Roux, F-75724 Paris Cedex 15, FranceAb...

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“…Crystallization and Data Collection-The protein was concentrated to 3-5 mg/ml in a buffer containing 50 mM Tris-HCl, pH 7.6, 250 mM NaCl, 2% glycerol, and 100 mM non-detergent Sulfobetaine 195 as a solubilizing agent (25). Crystals were obtained at 20°C in hanging drops containing 0.2-0.4% agarose, 2-3 l of the protein solution, and 2 l of the reservoir solution (28 -41% polyethylene glycol (PEG) 4000, 100 mM Tris-HCl, pH 7.0, 50 mM CaCl 2 , 10 mM ␤-mercaptoethanol).…”

Section: Methodsmentioning

confidence: 99%

The Crystal Structure of the Globular Head of Complement Protein C1q Provides a Basis for Its Versatile Recognition Properties

Gaboriaud¹,

Juanhuix

Gruez³

et al. 2003

Journal of Biological Chemistry

324

366

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C1q is a versatile recognition protein that binds to an amazing variety of immune and non-immune ligands and triggers activation of the classical pathway of complement. The crystal structure of the C1q globular domain responsible for its recognition properties has now been solved and refined to 1.9 Å of resolution. The structure reveals a compact, almost spherical heterotrimeric assembly held together mainly by non-polar interactions, with a Ca 2؉ ion bound at the top. The heterotrimeric assembly of the C1q globular domain appears to be a key factor of the versatile recognition properties of this protein. Plausible three-dimensional models of the C1q globular domain in complex with two of its physiological ligands, C-reactive protein and IgG, are proposed, highlighting two of the possible recognition modes of C1q. The C1q/human IgG1 model suggests a critical role for the hinge region of IgG and for the relative orientation of its Fab domain in C1q binding.Innate immunity involves a combination of cell-surface receptors and soluble proteins with the ability to recognize microbial pathogens and thereby to generate signals that both orientate subsequent adaptive immune responses and trigger effector mechanisms (1, 2). Most of these molecules are oligomeric and recognize molecular patterns on microorganisms (3). An archetypal molecule of this type is C1q, the recognition subunit of C1, the complex that triggers activation of the classical pathway of complement, a major element of innate immunity. C1q is a 460-kDa protein with the overall shape of a bouquet of flowers, comprising six heterotrimeric collagen-like triple helices that associate in their N-terminal half to form a "stalk," then diverge to form individual "stems", each terminating in a C-terminal heterotrimeric globular domain (4). It is well documented that most of the C1 complex ligands are recognized by these peripheral globular domains, or heads, of C1q, thus triggering activation of C1r and C1s, the proteases associated with C1q (5). It is also established that C1q binds to immune complexes containing IgG or IgM, but not to those having IgA, IgD, or IgE (6). The major C1q binding site on IgG has been mapped to the CH2 domain of the Fc portion of the molecule (7-9). Although C1q shows marked differences in its reactivity toward IgG subclasses, the reason for this selectivity is not known.C1q is traditionally known for its ability to bind antibodies. However, it recognizes an amazing variety of other ligands. These include certain bacteria, viruses, parasites, and mycoplasma (6, 10 -12), underscoring its role as an antibody-independent defense protein. C1q also binds to C-reactive protein (CRP) 1 when complexed with exposed phosphocholine residues on bacteria, providing a further means of host defense (13). C1q is also capable of recognizing aberrant structures from self. Thus, in addition to cellular debris and sub-cellular membranes (14), it is established that C1q binds to, and induces clearance of, apoptotic cells (15), thereby playing a major role ...

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A new additive for protein crystallization

Cited by 31 publications

References 15 publications

Inhibition of protein aggregation by zwitterionic polymer-based core-shell nanogels

Inhibition of protein aggregation by zwitterionic polymer-based core-shell nanogels

Interactions of non‐detergent sulfobetaines with early folding intermediates facilitate in vitro protein renaturation

The Crystal Structure of the Globular Head of Complement Protein C1q Provides a Basis for Its Versatile Recognition Properties

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