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1.
Detailed numerical study of the peak shapes of neutral analytes injected at high solvent strength in short reversed-phase liquid chromatography columns and comparison with experimental observations.
Pepermans, V, Chapel, S, Heinisch, S, Desmet, G
Journal of chromatography. A. 2021;:462078
Abstract
We report on a numerical investigation of the different steps in the development of the spatial concentration profiles developing along the axis of a liquid chromatography column when injecting large relative volumes (>10 to 20% of column volume) of analytes dissolved in a high solvent strength solvent band as can be encountered in the second dimension (2D) column of a two-dimensional liquid chromatography (2D-LC) system. More specifically, we made a detailed study of the different retention and the axial band broadening effects leading to the double-headed peak shapes or strongly fronting peaks that can be experimentally observed under certain conditions in 2D-LC. The establishment of these intricate peak profiles is discussed in all its fine, mechanistic details. The effect of the volume of the column, the volume and the shape of the sample band, the retention properties of the analyte and the band broadening experienced by the analytes and the sample solvent are investigated. A good agreement between the simulations and the experimental observations with caffeine and methylparaben injected in acetonitrile/water (ACN/H2O) mobile phase with different injection volumes is obtained. Save the difference in dwell volume, key features of experimental and simulated chromatograms agree within a few %. The simulations are also validated against a number of simple mathematical rules of thumb that can be established to predict the occurrence of a breakthrough fraction and estimate the amount of breakthrough.
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2.
Exploring Structure and Function of Redox Intermediates in [NiFe]-Hydrogenases by an Advanced Experimental Approach for Solvated, Lyophilized and Crystallized Metalloenzymes.
Lorent, C, Pelmenschikov, V, Frielingsdorf, S, Schoknecht, J, Caserta, G, Yoda, Y, Wang, H, Tamasaku, K, Lenz, O, Cramer, SP, et al
Angewandte Chemie (International ed. in English). 2021;(29):15854-15862
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Abstract
To study metalloenzymes in detail, we developed a new experimental setup allowing the controlled preparation of catalytic intermediates for characterization by various spectroscopic techniques. The in situ monitoring of redox transitions by infrared spectroscopy in enzyme lyophilizate, crystals, and solution during gas exchange in a wide temperature range can be accomplished as well. Two O2 -tolerant [NiFe]-hydrogenases were investigated as model systems. First, we utilized our platform to prepare highly concentrated hydrogenase lyophilizate in a paramagnetic state harboring a bridging hydride. This procedure proved beneficial for 57 Fe nuclear resonance vibrational spectroscopy and revealed, in combination with density functional theory calculations, the vibrational fingerprint of this catalytic intermediate. The same in situ IR setup, combined with resonance Raman spectroscopy, provided detailed insights into the redox chemistry of enzyme crystals, underlining the general necessity to complement X-ray crystallographic data with spectroscopic analyses.
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CompassR Yields Highly Organic-Solvent-Tolerant Enzymes through Recombination of Compatible Substitutions.
Cui, H, Jaeger, KE, Davari, MD, Schwaneberg, U
Chemistry (Weinheim an der Bergstrasse, Germany). 2021;(8):2789-2797
Abstract
The CompassR (computer-assisted recombination) rule enables, among beneficial substitutions, the identification of those that can be recombined in directed evolution. Herein, a recombination strategy is systematically investigated to minimize experimental efforts and maximize possible improvements. In total, 15 beneficial substitutions from Bacillus subtilis lipase A (BSLA), which improves resistance to the organic cosolvent 1,4-dioxane (DOX), were studied to compare two recombination strategies, the two-gene recombination process (2GenReP) and the in silico guided recombination process (InSiReP), employing CompassR. Remarkably, both strategies yielded a highly DOX-resistant variant, M4 (I12R/Y49R/E65H/N98R/K122E/L124K), with up to 14.6-fold improvement after screening of about 270 clones. M4 has a remarkably enhanced resistance in 60 % (v/v) acetone (6.0-fold), 30 % (v/v) ethanol (2.1-fold), and 60 % (v/v) methanol (2.4-fold) compared with wild-type BSLA. Molecular dynamics simulations revealed that attracting water molecules by charged surface substitutions is the main driver for increasing the DOX resistance of BSLA M4. Both strategies and obtained molecular knowledge can likely be used to improve the properties of other enzymes with a similar α/β-hydrolase fold.
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Creating Flavin Reductase Variants with Thermostable and Solvent-Tolerant Properties by Rational-Design Engineering.
Maenpuen, S, Pongsupasa, V, Pensook, W, Anuwan, P, Kraivisitkul, N, Pinthong, C, Phonbuppha, J, Luanloet, T, Wijma, HJ, Fraaije, MW, et al
Chembiochem : a European journal of chemical biology. 2020;(10):1481-1491
Abstract
We have employed computational approaches-FireProt and FRESCO-to predict thermostable variants of the reductase component (C1 ) of (4-hydroxyphenyl)acetate 3-hydroxylase. With the additional aid of experimental results, two C1 variants, A166L and A58P, were identified as thermotolerant enzymes, with thermostability improvements of 2.6-5.6 °C and increased catalytic efficiency of 2- to 3.5-fold. After heat treatment at 45 °C, both of the thermostable C1 variants remain active and generate reduced flavin mononucleotide (FMNH- ) for reactions catalyzed by bacterial luciferase and by the monooxygenase C2 more efficiently than the wild type (WT). In addition to thermotolerance, the A166L and A58P variants also exhibited solvent tolerance. Molecular dynamics (MD) simulations (6 ns) at 300-500 K indicated that mutation of A166 to L and of A58 to P resulted in structural changes with increased stabilization of hydrophobic interactions, and thus in improved thermostability. Our findings demonstrated that improvements in the thermostability of C1 enzyme can lead to broad-spectrum uses of C1 as a redox biocatalyst for future industrial applications.
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5.
Adduct under Field-A Qualitative Approach to Account for Solvent Effect on Hydrogen Bonding.
Shenderovich, IG, Denisov, GS
Molecules (Basel, Switzerland). 2020;(3)
Abstract
The location of a mobile proton in acid-base complexes in aprotic solvents can be predicted using a simplified Adduct under Field (AuF) approach, where solute-solvent effects on the geometry of hydrogen bond are simulated using a fictitious external electric field. The parameters of the field have been estimated using experimental data on acid-base complexes in CDF3/CDClF2. With some limitations, they can be applied to the chemically similar CHCl3 and CH2Cl2. The obtained data indicate that the solute-solvent effects are critically important regardless of the type of complexes. The temperature dependences of the strength and fluctuation rate of the field explain the behavior of experimentally measured parameters.
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Revisiting the Self-Assembly of Highly Aromatic Phenylalanine Homopeptides.
Mayans, E, Alemán, C
Molecules (Basel, Switzerland). 2020;(24)
Abstract
Diphenylalanine peptide (FF), which self-assembles into rigid tubular nanostructures, is a very short core recognition motif in Alzheimer's disease β-amyloid (Aβ) polypeptide. Moreover, the ability of the phenylalanine (F or Phe)-homopeptides to self-assemble into ordered nanostructures has been proved. Within this context it was shown that the assembly preferences of this family of compounds is altered by capping both the N- and C-termini using highly aromatic fluorenyl groups (i.e., fluorenyl-9-methoxycarbonyl and 9-fluorenylmethyl ester, named Fmoc and OFm, respectively). In this article the work performed in the field of the effect of the structure and incubation conditions on the morphology and polymorphism of short (from two to four amino acid residues) Phe-homopeptides is reviewed and accompanied by introducing some new results for completing the comparison. Special attention has been paid to the influence of solvent: co-solvent mixture used to solubilize the peptide, the peptide concentration and, in some cases, the temperature. More specifically, uncapped (FF, FFF, and FFFF), N-capped with Fmoc (Fmoc-FF, Fmoc-FFF, and Fmoc-FFFF), C-capped with OFm (FF-OFm), and doubly capped (Fmoc-FF-OFm, Fmoc-FFF-OFm, and Fmoc-FFFF-OFm) Phe-homopeptides have been re-measured. Although many of the experienced assembly conditions have been only revisited as they were previously reported, other experimental conditions have been examined by the first time in this work. In any case, pooling the effect of highly aromatic blocking groups in a single study, using a wide variety of experimental conditions, allows a perspective of how the disappearance of head-to-tail electrostatic interactions and the gradual increase in the amount of π-π stacking interactions, affects the morphology of the assemblies. Future technological applications of Phe-homopeptides can be envisaged by choosing the most appropriate self-assemble structure, defining not only the length of the peptide but also the amount and the position of fluorenyl capping groups.
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Protein Solvent Interaction: Transition of Protein-solvent Interaction Concept from Basic Research into Solvent Manipulation of Chromatography.
Arakawa, T, Kita, Y
Current protein & peptide science. 2019;(1):34-39
Abstract
Previously, we have reviewed in this journal (Arakawa, T., Kita, Y., Curr. Protein Pept. Sci., 15, 608-620, 2014) the interaction of arginine with proteins and various applications of this solvent additive in the area of protein formulations and downstream processes. In this special issue, we expand the concept of protein-solvent interaction into the analysis of the effects of solvent additives on various column chromatography, including mixed-mode chromatography. Earlier in our research, we have studied the interactions of such a variety of solvent additives as sugars, salts, amino acids, polymers and organic solvents with a variety of proteins, which resulted in mechanistic understanding on their protein stabilization and precipitation effects, the latter known as Hofmeister series. While such a study was then a pure academic research, rapid development of genetic engineering technologies and resultant biotechnologies made it a valuable knowledge in fully utilizing solvent additives in manipulation of protein solution, including column chromatography.
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Solvent Composition Effects on the Structural Properties of the Aβ42 Monomer from the 3D-RISM-KH Molecular Theory of Solvation.
Blinov, N, Wishart, DS, Kovalenko, A
The journal of physical chemistry. B. 2019;(11):2491-2506
Abstract
Structural characterization of amyloid (A)β peptides implicated in Alzheimer's disease is a challenging problem due to their intrinsically disordered nature and their high propensity for aggregation. Only limited information is currently available from experiments on conformational properties and aggregation pathways of the peptides in cellular environments. In silico modeling complements experimental information, providing atomistic insight into structure and dynamics of different Aβ species. All-atom explicit solvent molecular dynamics (MD) simulations with a properly selected force field can deliver reliable structural and dynamic information. In the case of intrinsically disordered Aβ peptides, enhanced sampling simulations beyond the nanosecond time scale are required to obtain statistically meaningful results even for simple solvent conditions. To overcome the challenges of conformational sampling in crowded cellular environments, alternative approaches have to be used, including postprocessing of MD data. In this study, we employ the statistical-mechanical, three-dimensional reference interaction site model with the Kovalenko-Hirata closure integral equation molecular theory of solvation to describe solvent composition effects on the conformational equilibrium in a structural ensemble of the Aβ42 (covering residues 1-42) monomer based on a statistical reweighting technique. The methodology enables a computationally efficient prediction on how different factors in the cellular environment, such as solvent composition, nonpolar solvation, and macromolecular crowding, affect the structural properties of the monomer. Similarities have been identified between changes in the structural ensemble caused by nonpolar solvation and crowded environments modeled by ionic solution with large negative ions. In particular, both solvent conditions reduce the random coil content and enhance the helical structure content of the monomer. In contrast to the previous studies, which reported increased α-helical content of peptides in crowded environments, this work attributes these structural features to the difference in solvent exposure of hydrophilic residues of the monomer for different secondary structure elements, rather than to (entropic) excluded volume effects.
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Four types of human platelet lysate, including one virally inactivated by solvent-detergent, can be used to propagate Wharton jelly mesenchymal stromal cells.
Chen, MS, Wang, TJ, Lin, HC, Burnouf, T
New biotechnology. 2019;:151-160
Abstract
There is accumulating experimental evidence that human platelet lysate (HPL) made from platelet concentrates can replace fetal bovine serum (FBS) as a xeno-free clinical-grade supplement of growth media to expand mesenchymal stromal cells (MSCs). However, uncertainties exist in regard to impacts that various manufacturing methods of HPL can exert on the expansion and differentiation capacity of MSCs. In particular, there is a need to evaluate the possibility of implementing virus-inactivation treatment during HPL production to ensure optimal safety of industrial HPL pools. Expired human platelet concentrates from four different donors were pooled and subjected to freeze-thaw cycles (-80/+37 °C), followed or not by serum-conversion by calcium chloride, heat-treatment at 56 °C for 30 min, or solvent-detergent (S/D) virus inactivation. The concentrations of total proteins, growth factors and fibrinogen, and the chemical compositions of the HPLs were characterized. The impact of HPL supplementation on the cell morphology, doubling time, immunophenotype and trilineage differentiation capacity of Wharton jelly MSCs (WJMSCs) were compared over five passages, using FBS as a control and normalizing the protein content. Data showed that WJMSCs expanded equally well, exhibited a typical fibroblast morphology, had short doubling times, maintained their immunophenotypes, and differentiated into chondrocyte, osteocyte, and adipocyte lineages in all HPL-supplemented media, all of which were more effective than FBS. In conclusion, we found minimal detectable impact of the HPL manufacturing process, including S/D virus inactivation, on the suitability of expanding WJMSCs in vitro.
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10.
Fast NMR method to probe solvent accessibility and disordered regions in proteins.
Faustino, AF, Barbosa, GM, Silva, M, Castanho, MARB, Da Poian, AT, Cabrita, EJ, Santos, NC, Almeida, FCL, Martins, IC
Scientific reports. 2019;(1):1647
Abstract
Understanding protein structure and dynamics, which govern key cellular processes, is crucial for basic and applied research. Intrinsically disordered protein (IDP) regions display multifunctionality via alternative transient conformations, being key players in disease mechanisms. IDP regions are abundant, namely in small viruses, allowing a large number of functions out of a small proteome. The relation between protein function and structure is thus now seen from a different perspective: as IDP regions enable transient structural arrangements, each conformer can play different roles within the cell. However, as IDP regions are hard and time-consuming to study via classical techniques (optimized for globular proteins with unique conformations), new methods are required. Here, employing the dengue virus (DENV) capsid (C) protein and the immunoglobulin-binding domain of streptococcal protein G, we describe a straightforward NMR method to differentiate the solvent accessibility of single amino acid N-H groups in structured and IDP regions. We also gain insights into DENV C flexible fold region biological activity. The method, based on minimal pH changes, uses the well-established 1H-15N HSQC pulse sequence and is easily implementable in current protein NMR routines. The data generated are simple to interpret, with this rapid approach being an useful first-choice IDPs characterization method.