State-of-the-Art Research in Biomolecular Crystals

A special issue of Crystals (ISSN 2073-4352). This special issue belongs to the section "Biomolecular Crystals".

Deadline for manuscript submissions: closed (31 March 2022) | Viewed by 23563

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Instituto de Química, Universidad Nacional Autónoma de México. Av. Universidad 3000, Cd.Mx. 04510, Mexico
Interests: protein crystals; biocrystals; crystal growth; protein crystallography; crystal chemistry; biomineralization; biomimetics; biological macromolecules
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Special Issue Information

Dear Colleagues,

Aiming to suggest a methodology for state-of-the art investigations on biomolecular crystals, the present Special Issue will be divided into four parts in a logical pathway to obtain the first suitable crystals for high-resolution X-ray crystallographic analysis, ranging from experimental methods to the use of different techniques of synchrotron radiation. The first part deals with the fundamentals of each crystallization method through different strategies and applications of biomolecular crystals based on physical and chemical approaches. The second part will present new approaches involved in more sophisticated techniques not only for growing protein crystals, but also for controlling the size and number of crystals through different techniques, from seeding techniques up to the use of electric and magnetic fields. The third part will be related to the gel-growth and counter-diffusion techniques such as spatiotemporal control of mass and heat to improve the crystallization process as well as the selection of cryo-protectant to obtain suitable protein crystals for X-ray diffraction. The structure of these biomolecules performed by X-ray diffraction will also be determined by SAXS in solution to combine different approaches where there are recalcitrant proteins to be crystallized. The envelop obtained by SAXS could be used to obtain the 3D structure via cryo-EM techniques, which will also be revised. Finally, the fourth part will be focused on the use of XFEL that has revolutionized the new concept of protein structure determination, but instead of using classical X-ray crystallography, the XFEL techniques will provide the structural data in a few minutes. These techniques will mold the new kind of future protein crystallographers trained in using novel software with big data recorded. Additionally, the tools for manipulating micro or nanocrystals are completely new and will also be included in this Special Issue.

Prof. Dr. Abel Moreno
Guest Editor

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Published Papers (12 papers)

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Editorial

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3 pages, 195 KiB  
Editorial
State-of-the Art Research in Biomolecular Crystals
by Abel Moreno
Crystals 2023, 13(1), 58; https://doi.org/10.3390/cryst13010058 - 29 Dec 2022
Viewed by 1121
Abstract
This special issue, State-of-the Art Investigations on Biomolecular Crystals, is focused on strategies to procure suitable crystals for high-resolution X-ray crystallographic investigations [...] Full article
(This article belongs to the Special Issue State-of-the-Art Research in Biomolecular Crystals)

Research

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12 pages, 4429 KiB  
Article
Synthesis, Molecular Docking, and Neuroprotective Effect of 2-Methylcinnamic Acid Amide in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)—An Induced Parkinson’s Disease Model
by Maya Chochkova, Rusi Rusew, Reni Kalfin, Lyubka Tancheva, Maria Lazarova, Hristina Sbirkova-Dimitrova, Andrey Popatanasov, Krasimira Tasheva, Boris Shivachev, Nejc Petek and Martin Štícha
Crystals 2022, 12(11), 1518; https://doi.org/10.3390/cryst12111518 - 26 Oct 2022
Cited by 1 | Viewed by 1717
Abstract
Parkinson’s disease (PD) has emerged as the second most common form of human neurodegenerative disorders. However, due to the severe side effects of the current antiparkinsonian drugs, the design of novel and safe compounds is a hot topic amongst the medicinal chemistry community. [...] Read more.
Parkinson’s disease (PD) has emerged as the second most common form of human neurodegenerative disorders. However, due to the severe side effects of the current antiparkinsonian drugs, the design of novel and safe compounds is a hot topic amongst the medicinal chemistry community. Herein, a convenient peptide method, TBTU (O-(benzotriazole-1-yl)-N,N,N′,N′-tetramethyluronium tetrafluoroborate), was used for the synthesis of the amide (E)-N-(2-methylcinnamoyl)-amantadine (CA(2-Me)-Am; 3)) derived from amantadine and 2-methylcinnamic acid. The obtained hybrid was studied for its antiparkinsonian activity in an experimental model of PD induced by MPTP. Mice (C57BL/6,male, 8 weeks old) were divided into four groups as follows: (1) the control, treated with normal saline (i.p.) for 12 consecutive days; (2) MPTP (30 mg/kg/day, i.p.), applied daily for 5 consecutive days; (3) MPTP + CA(2-Me)-Am, applied for 12 consecutive days, 5 days simultaneously with MPTP and 7 days after MPTP; (4) CA(2-Me)-Am +oleanoic acid (OA), applied daily for 12 consecutive days. Neurobehavioral parameters in all experimental groups of mice were evaluated by rotarod test and passive avoidance test. Our experimental data showed that CA(2-Me)-Am in parkinsonian mice significantly restored memory performance, while neuromuscular coordination approached the control level, indicating the ameliorating effects of the new compound. In conclusion, the newly synthesized hybrid might be a promising agent for treating motor disturbances and cognitive impairment in experimental PD. Full article
(This article belongs to the Special Issue State-of-the-Art Research in Biomolecular Crystals)
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11 pages, 1669 KiB  
Article
The Effect of DNA from Escherichia Coli at High and Low CO2 Concentrations on the Shape and Form of Crystal-line Silica-Carbonates of Barium (II)
by Cesia D. Pérez-Aguilar, Selene R. Islas, Abel Moreno and Mayra Cuéllar-Cruz
Crystals 2022, 12(8), 1147; https://doi.org/10.3390/cryst12081147 - 15 Aug 2022
Cited by 1 | Viewed by 1341
Abstract
The synthesis of nucleic acids in the Precambrian era marked the start of life, with DNA being the molecule in which the genetic information has been conserved ever since. After studying the DNA of different organisms for several decades, we now know that [...] Read more.
The synthesis of nucleic acids in the Precambrian era marked the start of life, with DNA being the molecule in which the genetic information has been conserved ever since. After studying the DNA of different organisms for several decades, we now know that cell size and cellular differentiation are influenced by DNA concentration and environmental conditions. However, we still need to find out the minimum required concentration of DNA in the pioneer cell to control the resulting morphology. In order to do this, the present research aims to evaluate the influence of the DNA concentration on the morphology adopted by biomorphs (barium silica-carbonates) under two synthesis conditions: one emulating the Precambrian era and one emulating the present era. The morphology of the synthetized biomorphs was assessed through scanning electron microscopy (SEM). The chemical composition and the crystalline structure were determined through Raman and IR spectroscopy. Our results showed that DNA, even at relatively low levels, affects the morphology of the biomorph structure. They also indicated that, even at the low DNA concentration prevailing during the synthesis of the first DNA biomolecules existing in the primitive era, these biomolecules influenced the morphology of the inorganic structure that lodged it. On the other hand, this also allows us to infer that, once the DNA was synthetized in the Precambrian era, it was definitely responsible for generating, conserving, and directing the morphology of all organisms up to the present day. Full article
(This article belongs to the Special Issue State-of-the-Art Research in Biomolecular Crystals)
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12 pages, 1593 KiB  
Article
Studies on the Crystal Forms of Istradefylline: Structure, Solubility, and Dissolution Profile
by Yiyun Wang, Youwei Xu, Zhonghui Zheng, Min Xue, Zihui Meng, Zhibin Xu, Jiarong Li and Qing Lin
Crystals 2022, 12(7), 917; https://doi.org/10.3390/cryst12070917 - 28 Jun 2022
Cited by 2 | Viewed by 2333
Abstract
Istradefylline as a selective adenosine A2A-receptor antagonist is clinically used to treat Parkinson’s disease and improve dyskinesia in its early stages. However, its crystal form, as an important factor in the efficacy of the drug, is rarely studied. Herein, three [...] Read more.
Istradefylline as a selective adenosine A2A-receptor antagonist is clinically used to treat Parkinson’s disease and improve dyskinesia in its early stages. However, its crystal form, as an important factor in the efficacy of the drug, is rarely studied. Herein, three kinds of crystal forms of istradefylline prepared from ethanol (form I), methanol (form II), and acetonitrile (form III) are reported by use of a crystal engineering strategy. These three crystal forms were characterized and made into tablets for dissolution testing. Both the solubility and the dissolution rates were also determined. The dissolution rate of form I and form III is significantly higher than form II at pH 1.2 (87.1%, 58.2%, and 87.7% for form I, form II, and form III, respectively), pH 4.5 (88.1%, 58.9%, and 87.1% for form I, form II, and form III, respectively) and pH 6.8 (87.5%, 58.2%, and 86.0% for form I, form II, and form III, respectively) at 60 min. Considering the prepared solution and the proper dissolution profile, form I is anticipated to possess promising absorption for bioavailability. Full article
(This article belongs to the Special Issue State-of-the-Art Research in Biomolecular Crystals)
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15 pages, 3579 KiB  
Article
Through Diffusion Measurements of Molecules to a Numerical Model for Protein Crystallization in Viscous Polyethylene Glycol Solution
by Hiroaki Tanaka, Rei Utata, Keiko Tsuganezawa, Sachiko Takahashi and Akiko Tanaka
Crystals 2022, 12(7), 881; https://doi.org/10.3390/cryst12070881 - 21 Jun 2022
Cited by 1 | Viewed by 1806
Abstract
Protein crystallography has become a popular method for biochemists, but obtaining high-quality protein crystals for precise structural analysis and larger ones for neutron analysis requires further technical progress. Many studies have noted the importance of solvent viscosity for the probability of crystal nucleation [...] Read more.
Protein crystallography has become a popular method for biochemists, but obtaining high-quality protein crystals for precise structural analysis and larger ones for neutron analysis requires further technical progress. Many studies have noted the importance of solvent viscosity for the probability of crystal nucleation and for mass transportation; therefore, in this paper, we have reported on experimental results and simulation studies regarding the use of viscous polyethylene glycol (PEG) solvents for protein crystals. We investigated the diffusion rates of proteins, peptides, and small molecules in viscous PEG solvents using fluorescence correlation spectroscopy. In high-molecular-weight PEG solutions (molecular weights: 10,000 and 20,000), solute diffusion showed deviations, with a faster diffusion than that estimated by the Stokes–Einstein equation. We showed that the extent of the deviation depends on the difference between the molecular sizes of the solute and PEG solvent, and succeeded in creating equations to predict diffusion coefficients in viscous PEG solutions. Using these equations, we have developed a new numerical model of 1D diffusion processes of proteins and precipitants in a counter-diffusion chamber during crystallization processes. Examples of the application of anomalous diffusion in counter-diffusion crystallization are shown by the growth of lysozyme crystals. Full article
(This article belongs to the Special Issue State-of-the-Art Research in Biomolecular Crystals)
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14 pages, 8795 KiB  
Article
Crystal Breakage Due to Combined Normal and Shear Loading
by Benjamin Radel, Marco Gleiß and Hermann Nirschl
Crystals 2022, 12(5), 644; https://doi.org/10.3390/cryst12050644 - 30 Apr 2022
Cited by 4 | Viewed by 1630
Abstract
Combined normal and shear stress on particles occurs in many devices for solid–liquid separation. Protein crystals are much more fragile compared to conventional crystals because of their high water content. Therefore, unwanted crystal breakage is to be expected in the processing of such [...] Read more.
Combined normal and shear stress on particles occurs in many devices for solid–liquid separation. Protein crystals are much more fragile compared to conventional crystals because of their high water content. Therefore, unwanted crystal breakage is to be expected in the processing of such materials. The influence of pressure and shearing has been investigated individually in the past. To analyze the influence of combined shear and normal stress on protein crystals, a modified shear cell for a ring shear tester is used. This device allows one to accurately vary the normal and shear stress on moist crystals in a saturated particle bed. Analyzing the protein crystals in a moist state is important because the mechanical properties change significantly after drying. The results show a big influence of the applied normal stress on crystal breakage while shearing. Higher normal loading leads to a much bigger comminution. The shear velocity, however, has a comparatively negligible influence. Full article
(This article belongs to the Special Issue State-of-the-Art Research in Biomolecular Crystals)
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11 pages, 2883 KiB  
Article
The Effect of Controlled Mixing on ROY Polymorphism
by Margot Van Nerom, Pierre Gelin, Mehrnaz Hashemiesfahan, Wim De Malsche, James F. Lutsko, Dominique Maes and Quentin Galand
Crystals 2022, 12(5), 577; https://doi.org/10.3390/cryst12050577 - 20 Apr 2022
Cited by 5 | Viewed by 1848
Abstract
We report the investigation of various experimental conditions and their influence on polymorphism of 5-methyl-2-[(2-nitrophenyl)amino]-3-thiophenecarbonitrile, commonly known as ROY. These conditions include an in-house-developed microfluidic chip with controlled mixing of parallel flows. We observed that different ROY concentrations and different solvent to antisolvent [...] Read more.
We report the investigation of various experimental conditions and their influence on polymorphism of 5-methyl-2-[(2-nitrophenyl)amino]-3-thiophenecarbonitrile, commonly known as ROY. These conditions include an in-house-developed microfluidic chip with controlled mixing of parallel flows. We observed that different ROY concentrations and different solvent to antisolvent ratios naturally favored different polymorphs. Nonetheless, identical samples prepared with different mixing methods, such as rotation and magnetic stirring, consistently led to the formation of different polymorphs. A fourth parameter, namely the confinement of the sample, was also considered. Untangling all those parameters and their influences on polymorphism called for an experimental setup allowing all four to be controlled accurately. To that end, we developed a novel customized microfluidic setup allowing reproducible and controlled mixing conditions. Two parallel flows of antisolvent and ROY dissolved in solvent were infused into a transparent microchannel. Next, slow and progressive mixing could be obtained by molecular diffusion. Additionally, the microfluidic chip was equipped with a piezoceramic element, allowing the implementation of various mixing rates by acoustic mixing. With this device, we demonstrated the importance of parameters other than concentration on the polymorphism of ROY. Full article
(This article belongs to the Special Issue State-of-the-Art Research in Biomolecular Crystals)
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15 pages, 3833 KiB  
Article
Structure-Based Modeling of the Mechanical Behavior of Cross-Linked Enzyme Crystals
by Marta Kubiak, Ingo Kampen and Carsten Schilde
Crystals 2022, 12(4), 441; https://doi.org/10.3390/cryst12040441 - 22 Mar 2022
Cited by 5 | Viewed by 2022
Abstract
Because of their high volumetric catalytic activity, in addition to their high chemical and thermal resistances, enzymes in the form of protein crystals are an excellent choice for application as immobilized biocatalysts. However, mechanical stability is a requirement for the processability of immobilisates, [...] Read more.
Because of their high volumetric catalytic activity, in addition to their high chemical and thermal resistances, enzymes in the form of protein crystals are an excellent choice for application as immobilized biocatalysts. However, mechanical stability is a requirement for the processability of immobilisates, in addition to the protein crystals retaining their enzymatic activity, and this is closely related to the crystal structure. In this study, the influence of protein engineering on the mechanical stability of cross-linked enzyme crystals (CLECs) was investigated using a genetically modified model protein in which additionally cysteines were introduced on the protein surface for targeted cross-linking. The results showed that the mechanical stability of crystals of the mutant proteins in the native form was decreased compared to native wild-type crystals. However, specific cross-linking of the introduced amino acid residues in the mutant proteins resulted in their increased mechanical stability compared to wild-type CLECs. In order to determine the correlation between the crystal structure and the resulting mechanical properties of CLECs to enable targeted cross-linking, a previously developed model was revised and then used for the two model proteins. This model can explain the mechanically investigated relationships, such as the anisotropic crystal behavior and the influence of a linker or mutation on the micromechanical properties and, hence, can be helpful for the tailor-made production of CLECs. Full article
(This article belongs to the Special Issue State-of-the-Art Research in Biomolecular Crystals)
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21 pages, 31906 KiB  
Article
Stabilizing DNA–Protein Co-Crystals via Intra-Crystal Chemical Ligation of the DNA
by Abigail R. Orun, Sara Dmytriw, Ananya Vajapayajula and Christopher D. Snow
Crystals 2022, 12(1), 49; https://doi.org/10.3390/cryst12010049 - 30 Dec 2021
Cited by 6 | Viewed by 2869 | Correction
Abstract
Protein and DNA co-crystals are most commonly prepared to reveal structural and functional details of DNA-binding proteins when subjected to X-ray diffraction. However, biomolecular crystals are notoriously unstable in solution conditions other than their native growth solution. To achieve greater application utility beyond [...] Read more.
Protein and DNA co-crystals are most commonly prepared to reveal structural and functional details of DNA-binding proteins when subjected to X-ray diffraction. However, biomolecular crystals are notoriously unstable in solution conditions other than their native growth solution. To achieve greater application utility beyond structural biology, biomolecular crystals should be made robust against harsh conditions. To overcome this challenge, we optimized chemical DNA ligation within a co-crystal. Co-crystals from two distinct DNA-binding proteins underwent DNA ligation with the carbodiimide crosslinking agent 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) under various optimization conditions: 5′ vs. 3′ terminal phosphate, EDC concentration, EDC incubation time, and repeated EDC dose. This crosslinking and DNA ligation route did not destroy crystal diffraction. In fact, the ligation of DNA across the DNA–DNA junctions was clearly revealed via X-ray diffraction structure determination. Furthermore, crystal macrostructure was fortified. Neither the loss of counterions in pure water, nor incubation in blood serum, nor incubation at low pH (2.0 or 4.5) led to apparent crystal degradation. These findings motivate the use of crosslinked biomolecular co-crystals for purposes beyond structural biology, including biomedical applications. Full article
(This article belongs to the Special Issue State-of-the-Art Research in Biomolecular Crystals)
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16 pages, 42859 KiB  
Article
A New L-Proline Amide Hydrolase with Potential Application within the Amidase Process
by Sergio Martinez-Rodríguez, Rafael Contreras-Montoya, Jesús M. Torres, Luis Álvarez de Cienfuegos and Jose Antonio Gavira
Crystals 2022, 12(1), 18; https://doi.org/10.3390/cryst12010018 - 23 Dec 2021
Cited by 1 | Viewed by 2941
Abstract
L-proline amide hydrolase (PAH, EC 3.5.1.101) is a barely described enzyme belonging to the peptidase S33 family, and is highly similar to prolyl aminopeptidases (PAP, EC. 3.4.11.5). Besides being an S-stereoselective character towards piperidine-based carboxamides, this enzyme also hydrolyses different L-amino acid [...] Read more.
L-proline amide hydrolase (PAH, EC 3.5.1.101) is a barely described enzyme belonging to the peptidase S33 family, and is highly similar to prolyl aminopeptidases (PAP, EC. 3.4.11.5). Besides being an S-stereoselective character towards piperidine-based carboxamides, this enzyme also hydrolyses different L-amino acid amides, turning it into a potential biocatalyst within the Amidase Process. In this work, we report the characterization of L-proline amide hydrolase from Pseudomonas syringae (PsyPAH) together with the first X-ray structure for this class of L-amino acid amidases. Recombinant PsyPAH showed optimal conditions at pH 7.0 and 35 °C, with an apparent thermal melting temperature of 46 °C. The enzyme behaved as a monomer at the optimal pH. The L-enantioselective hydrolytic activity towards different canonical and non-canonical amino-acid amides was confirmed. Structural analysis suggests key residues in the enzymatic activity. Full article
(This article belongs to the Special Issue State-of-the-Art Research in Biomolecular Crystals)
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20 pages, 12444 KiB  
Article
Shifts in Backbone Conformation of Acetylcholinesterases upon Binding of Covalent Inhibitors, Reversible Ligands and Substrates
by Zoran Radić
Crystals 2021, 11(12), 1557; https://doi.org/10.3390/cryst11121557 - 14 Dec 2021
Cited by 6 | Viewed by 2083
Abstract
The influence of ligand binding to human, mouse and Torpedo californica acetylcholinesterase (EC 3.1.1.7; AChE) backbone structures is analyzed in a pairwise fashion by comparison with X-ray structures of unliganded AChEs. Both complexes with reversible ligands (substrates and inhibitors) as well as covalently [...] Read more.
The influence of ligand binding to human, mouse and Torpedo californica acetylcholinesterase (EC 3.1.1.7; AChE) backbone structures is analyzed in a pairwise fashion by comparison with X-ray structures of unliganded AChEs. Both complexes with reversible ligands (substrates and inhibitors) as well as covalently interacting ligands leading to the formation of covalent AChE conjugates of tetrahedral and of trigonal-planar geometries are considered. The acyl pocket loop (AP loop) in the AChE backbone is recognized as the conformationally most adaptive, but not necessarily sterically exclusive, structural element. Conformational changes of the centrally located AP loop coincide with shifts in C-terminal α-helical positions, revealing interacting components for a potential allosteric interaction within the AChE backbone. The stabilizing power of the aromatic choline binding site, with the potential to attract and pull fitting entities covalently tethered to the active Ser, is recognized. Consequently, the pull can promote catalytic reactions or relieve steric pressure within the impacted space of the AChE active center gorge. These dynamic properties of the AChE backbone inferred from the analysis of static X-ray structures contribute towards a better understanding of the molecular template important in the structure-based design of therapeutically active molecules, including AChE inhibitors as well as reactivators of conjugated, inactive AChE. Full article
(This article belongs to the Special Issue State-of-the-Art Research in Biomolecular Crystals)
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5 pages, 707 KiB  
Correction
Correction: Orun et al. Stabilizing DNA–Protein Co-Crystals via Intra-Crystal Chemical Ligation of the DNA. Crystals 2022, 12, 49
by Abigail R. Orun, Sara Dmytriw, Ananya Vajapayajula and Christopher D. Snow
Crystals 2023, 13(4), 675; https://doi.org/10.3390/cryst13040675 - 14 Apr 2023
Cited by 1 | Viewed by 818
Abstract
In the publication [...] Full article
(This article belongs to the Special Issue State-of-the-Art Research in Biomolecular Crystals)
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