Protein crystallization in short-peptide supramolecular hydrogels: a versatile strategy towards biotechnological composite materials

Conejero-Muriel, Mayte; Contreras‐Montoya, Rafael; Díaz‐Mochón, Juan José; Cienfuegos, Luı́s Álvarez de; Gavira, José A.

doi:10.1039/c5ce00850f

Cited by 23 publications

(28 citation statements)

References 36 publications

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“…As already described for other dipeptide hydrogels [16,17], and with hydrogels in general [4], Fmoc-MF hydrogels grown with lysozyme crystals had very high quality, as shown by several crystallographic indicators (resolution limit, mosaicity, B-factor, and I/σ), and compared well with those obtained in agarose as reference ( Table 2 and Table S1). The diffraction data quality from lysozyme crystals was independent of the hydrogel concentration, although some improvement was observed particularly for one of the crystals grown in 0.75% (w/v) being the best one of this series, as was shown not only for the highest resolution limit, 1.15 Å resolution, but also from the lower values of the B-factor and mosaicity, which corresponded to a 20% and 40% improvement, respectively, respect to the average values.…”

Section: Agarosesupporting

confidence: 72%

“…We used the counter-diffusion technique in two-layer configurations to run the crystallization experiments. In order to charge the hydrogel, the protein solution at twice the final required concentration was poured on top of the gel layer, and left to diffuse into the hydrogel for one week following the previously detailed protocol [17]. Then, the equilibrated protein solution was substituted by the precipitant cocktail, and the experiments were kept at 295 K using incubators.…”

Section: Crystallization Experimentsmentioning

confidence: 99%

“…Lately, we have been exploring the incorporation of short peptide supramolecular (SPS) hydrogels to extend the pool of gels to novel biocompatible and biodegradable materials with broad biological and technological applications [14,15]. The use of these easily and broadly functionalizable compounds significantly expands the field of protein crystallization, allowing the study of the influence of the peptide chemical composition and chirality in crystallogenesis [16,17]. Besides, the incorporation of the gel fibers does not influence the three-dimensional (3D) structure, as previously observed with many other hydrogels; eventually, it influences the packing, generating new polymorphs [16].…”

Section: Introductionmentioning

confidence: 99%

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Extending the pool of compatible peptide hydrogels for protein crystallization

et al. 2019

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Short-peptide supramolecular (SPS) hydrogels are a class of materials that have been found to be useful for (bio)technological applications thanks to their biocompatible nature. Among the advantages reported for these peptides, their economic affordability and easy functionalization or modulation have turned them into excellent candidates for the development of functional biomaterials. We have recently demonstrated that SPS hydrogels can be used to produce high-quality protein crystals, improve their properties, or incorporate relevant materials within the crystals. In this work, we prove that hydrogels based on methionine and tyrosine are also good candidates for growing high-quality crystals of the three model proteins: lysozyme, glucose isomerase, and thaumatin.

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Section: Agarosesupporting

confidence: 72%

Section: Crystallization Experimentsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Extending the pool of compatible peptide hydrogels for protein crystallization

et al. 2019

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“…The growth of new polymorphs in gel media has been previously reported for proteins, when using peptide hydrogels [45,46], and for inorganic crystals grown in agarose The small isometric crystal-approximately 400 µm-and the needle crystals were cryoprotected, and X-ray data were collected at synchrotron source. The biggest crystals correspond to the already reported P4 3 2 1 2 space group [42][43][44], while the needles belong to the P2 1 2 1 2 1 space group (see Table 2 for details).…”

Section: Exploring the Low Supersaturation Regime Using Agarose Gelsmentioning

confidence: 91%

Tuning Transport Phenomena in Agarose Gels for the Control of Protein Nucleation Density and Crystal Form

et al. 2021

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Agarose gels provide the ideal environment for studying the nucleation step of complex biomacromolecules under diffusion-controlled conditions. In the present paper, we characterized the influence of agarose on the nucleation of three model proteins, i.e., lysozyme, insulin, and proteinase K, as a function of the agarose concentration using a batch method set-up inside flat capillaries. By using this set-up, we were able to directly count the number of crystals in a given volume and correlate it with the amount of agarose and with the average crystal size. We also studied the crystallization behavior of proteinase K with free-interface diffusion so that batch conditions were achieved through slow diffusion of the precipitant. Thanks to the control over the protein mass transport imposed by the network, a previously unknown crystal form, P212121, was obtained, and the three-dimensional structure was determined at a 1.6 Å resolution. Overall, the versatility of agarose gels makes them ideal candidates for the preparation of microcrystalline suspensions of biopharmaceuticals with precise and reproducible crystal attributes or for the exploration of the existence of different polymorphs.

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“…Finally, (4) the chip is sealed at each end with Crystal Clear tape (yellow). study (Conejero-Muriel et al, 2015). The initial experimental conditions are summarized in Table 1.…”

Section: Figurementioning

confidence: 99%

Attaining atomic resolution from in situ data collection at room temperature using counter-diffusion-based low-cost microchips

Gavira

Rodríguez-Ruiz

Martínez‐Rodríguez

et al. 2020

Acta Cryst Sect D Struct Biol

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Sample handling and manipulation for cryoprotection currently remain critical factors in X-ray structural determination. While several microchips for macromolecular crystallization have been proposed during the last two decades to partially overcome crystal-manipulation issues, increased background noise originating from the scattering of chip-fabrication materials has so far limited the attainable resolution of diffraction data. Here, the conception and use of low-cost, X-ray-transparent microchips for in situ crystallization and direct data collection, and structure determination at atomic resolution close to 1.0 Å, is presented. The chips are fabricated by a combination of either OSTEMER and Kapton or OSTEMER and Mylar materials for the implementation of counter-diffusion crystallization experiments. Both materials produce a sufficiently low scattering background to permit atomic resolution diffraction data collection at room temperature and the generation of 3D structural models of the tested model proteins lysozyme, thaumatin and glucose isomerase. Although the high symmetry of the three model protein crystals produced almost complete data sets at high resolution, the potential of in-line data merging and scaling of the multiple crystals grown along the microfluidic channels is also presented and discussed.

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Protein crystallization in short-peptide supramolecular hydrogels: a versatile strategy towards biotechnological composite materials

Cited by 23 publications

References 36 publications

Extending the pool of compatible peptide hydrogels for protein crystallization

Extending the pool of compatible peptide hydrogels for protein crystallization

Tuning Transport Phenomena in Agarose Gels for the Control of Protein Nucleation Density and Crystal Form

Attaining atomic resolution from in situ data collection at room temperature using counter-diffusion-based low-cost microchips

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