scholarly journals Protein Crystallization in a Microfluidic Contactor with Nafion®117 Membranes

Membranes ◽  
2021 ◽  
Vol 11 (8) ◽  
pp. 549
Author(s):  
M. Polino ◽  
H. S. Rho ◽  
M. P. Pina ◽  
R. Mallada ◽  
A. L. Carvalho ◽  
...  
Keyword(s):  
Protein Crystallization ◽  
Microfluidic System ◽  
Positive Influence ◽  
Empirical Science ◽  
Membrane Diffusion ◽  
X Ray ◽  
Egg White Lysozyme ◽  
Multiple Protein ◽  
Derivatizing Agent ◽  
Model Protein

Protein crystallization still remains mostly an empirical science, as the production of crystals with the required quality for X-ray analysis is dependent on the intensive screening of the best protein crystallization and crystal’s derivatization conditions. Herein, this demanding step was addressed by the development of a high-throughput and low-budget microfluidic platform consisting of an ion exchange membrane (117 Nafion® membrane) sandwiched between a channel layer (stripping phase compartment) and a wells layer (feed phase compartment) forming 75 independent micro-contactors. This microfluidic device allows for a simultaneous and independent screening of multiple protein crystallization and crystal derivatization conditions, using Hen Egg White Lysozyme (HEWL) as the model protein and Hg2+ as the derivatizing agent. This microdevice offers well-regulated crystallization and subsequent crystal derivatization processes based on the controlled transport of water and ions provided by the 117 Nafion® membrane. Diffusion coefficients of water and the derivatizing agent (Hg2+) were evaluated, showing the positive influence of the protein drop volume on the number of crystals and crystal size. This microfluidic system allowed for crystals with good structural stability and high X-ray diffraction quality and, thus, it is regarded as an efficient tool that may contribute to the enhancement of the proteins’ crystals structural resolution.

Potential use of ultrasound to promote protein crystallization

2010 ◽  
Vol 43 (6) ◽  
pp. 1419-1425 ◽  
Author(s):  
Rosa Crespo ◽  
Pedro M. Martins ◽  
Luís Gales ◽  
Fernando Rocha ◽  
Ana M. Damas
Keyword(s):  
Protein Crystallization ◽  
Crystal Formation ◽  
Data Sets ◽  
Resolution Limit ◽  
X Ray Diffraction ◽  
Promoting Effect ◽  
X Ray ◽  
X Ray Crystallography ◽  
Egg White Lysozyme ◽  
Crystallization Experiments

This work shows promising applications of ultrasound in promoting protein crystallization, which is important for structure determination by X-ray crystallography. It was observed that ultrasound can be used as a nucleation promoter as it decreases the energy barrier for crystal formation. Crystallization experiments on egg-white lysozyme were carried out with and without ultrasonic irradiation using commercial crystallization plates placed in temperature-controlled water baths. The nucleation-promoting effect introduced by ultrasound is illustrated by the reduction of the metastable zone width, as measured by the isothermal microbatch technique. The same effect was confirmed by the increased number of conditions leading to the formation of crystals when vapour diffusion techniques were carried out in the presence of ultrasound. By inducing faster nucleation, ultrasound leads to protein crystals grown at low supersaturation levels, which are known to have better diffraction properties. In fact, X-ray diffraction data sets collected using 13 lysozyme crystals (seven grown with ultrasound and six without) show an average 0.1 Å improvement in the resolution limit when ultrasound was used (p< 0.10). Besides the immediate application of ultrasound in nucleation promotion, the preliminary diffraction results also suggest a promising application in crystal quality enhancement.

scholarly journals Unusual Structural Features in the Adduct of Dirhodium Tetraacetate with Lysozyme

2021 ◽  
Vol 22 (3) ◽  
pp. 1496
Author(s):  
Domenico Loreto ◽  
Giarita Ferraro ◽  
Antonello Merlino
Keyword(s):  
Anticancer Agents ◽  
Structural Features ◽  
Side Chain ◽  
Side Chains ◽  
Valuable Insight ◽  
Experimental Conditions ◽  
Egg White Lysozyme ◽  
Potential Applications ◽  
Model Protein ◽  
Hen Egg White

The structures of the adducts formed upon reaction of the cytotoxic paddlewheel dirhodium complex [Rh2(μ-O2CCH3)4] with the model protein hen egg white lysozyme (HEWL) under different experimental conditions are reported. Results indicate that [Rh2(μ-O2CCH3)4] extensively reacts with HEWL:it in part breaks down, at variance with what happens in reactions with other proteins. A Rh center coordinates the side chains of Arg14 and His15. Dimeric Rh–Rh units with Rh–Rh distances between 2.3 and 2.5 Å are bound to the side chains of Asp18, Asp101, Asn93, and Lys96, while a dirhodium unit with a Rh–Rh distance of 3.2–3.4 Å binds the C-terminal carboxylate and the side chain of Lys13 at the interface between two symmetry-related molecules. An additional monometallic fragment binds the side chain of Lys33. These data, which are supported by replicated structural determinations, shed light on the reactivity of dirhodium tetracarboxylates with proteins, providing useful information for the design of new Rh-containing biomaterials with an array of potential applications in the field of catalysis or of medicinal chemistry and valuable insight into the mechanism of action of these potential anticancer agents.

Preliminary X-ray investigation of duck egg-white lysozyme II

1972 ◽  
Vol 71 (3) ◽  
pp. 815-817 ◽  
Author(s):  
J. Berthou ◽  
A. Laurent ◽  
P. Jolle`s
Keyword(s):  
Egg White ◽  
X Ray ◽  
Egg White Lysozyme ◽  
Duck Egg

Oxaliplatin vs. cisplatin: competition experiments on their binding to lysozyme

2015 ◽  
Vol 44 (22) ◽  
pp. 10392-10398 ◽  
Author(s):  
Daniela Marasco ◽  
Luigi Messori ◽  
Tiziano Marzo ◽  
Antonello Merlino
Keyword(s):  
Egg White ◽  
Hen Egg White Lysozyme ◽  
Egg White Lysozyme ◽  
Model Protein ◽  
Hen Egg White ◽  
Hen Egg

The model protein hen egg white lysozyme was challenged with oxaliplatin and cisplatin.

Mapping the protein-binding sites for iridium(iii)-based CO-releasing molecules

2016 ◽  
Vol 45 (30) ◽  
pp. 12206-12214 ◽  
Author(s):  
Marco Caterino ◽  
Ariel A. Petruk ◽  
Alessandro Vergara ◽  
Giarita Ferraro ◽  
Daniela Marasco ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Protein Binding ◽  
Binding Sites ◽  
Raman Microspectroscopy ◽  
Rnase A ◽  
Protein Binding Sites ◽  
X Ray ◽  
X Ray Crystallography ◽  
Model Protein ◽  
Circular Dichroïsm

Mass spectrometry, Raman microspectroscopy, circular dichroism and X-ray crystallography have been used to investigate the reaction of CO-releasing molecule Cs2IrCl5CO with the model protein RNase A.

scholarly journals Surface Engineering of Top7 to Facilitate Structure Determination

2022 ◽  
Vol 23 (2) ◽  
pp. 701
Author(s):  
Yuki Ito ◽  
Takuya Araki ◽  
Shota Shiga ◽  
Hiroyuki Konno ◽  
Koki Makabe
Keyword(s):  
Structure Determination ◽  
Surface Engineering ◽  
De Novo ◽  
Crystal Packing ◽  
Structure Model ◽  
X Ray ◽  
Hexahistidine Tag ◽  
Model Protein ◽  
Β Sheet ◽  
Packing Interactions

Top7 is a de novo designed protein whose amino acid sequence has no evolutional trace. Such a property makes Top7 a suitable scaffold for studying the pure nature of protein and protein engineering applications. To use Top7 as an engineering scaffold, we initially attempted structure determination and found that crystals of our construct, which lacked the terminal hexahistidine tag, showed weak diffraction in X-ray structure determination. Thus, we decided to introduce surface residue mutations to facilitate crystal structure determination. The resulting surface mutants, Top7sm1 and Top7sm2, crystallized easily and diffracted to the resolution around 1.7 Å. Despite the improved data, we could not finalize the structures due to high R values. Although we could not identify the origin of the high R values of the surface mutants, we found that all the structures shared common packing architecture with consecutive intermolecular β-sheet formation aligned in one direction. Thus, we mutated the intermolecular interface to disrupt the intermolecular β-sheet formation, expecting to form a new crystal packing. The resulting mutant, Top7sm2-I68R, formed new crystal packing interactions as intended and diffracted to the resolution of 1.4 Å. The surface mutations contributed to crystal packing and high resolution. We finalized the structure model with the R/Rfree values of 0.20/0.24. Top7sm2-I68R can be a useful model protein due to its convenient structure determination.

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