Probing multimodal ligand binding regions on ubiquitin using nuclear magnetic resonance, chromatography, and molecular dynamics simulations

Holstein, Melissa; Chung, Wai Keen; Parimal, Siddharth; Freed, Alexander S.; Barquera, Blanca; McCallum, Scott A.; Cramer, Steven M.

doi:10.1016/j.chroma.2011.12.101

Cited by 30 publications

(42 citation statements)

References 47 publications

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“…The chromatographic retention behavior of the charge Fab variants was obtained under linear gradient conditions in weak CEX (CM Sepharose Fast Flow) and multimodal (Capto MMC) chromatographic systems (Ligands presented in Table ). As shown in Figure B, the elution salt concentrations were significantly higher in the multimodal system as compared to the CEX material reflecting the stronger multimodal binding in agreement with previous results in the literature (Chung et al, ; Holstein et al, ). Furthermore, all charge variants exhibited decreased retention in both resin systems relative to the wild type.…”

Section: Resultssupporting

confidence: 91%

Investigation of protein selectivity in multimodal chromatography using in silico designed Fab fragment variants

Karkov

Krogh

Woo

et al. 2015

Biotech & Bioengineering

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In this study, a unique set of antibody Fab fragments was designed in silico and produced to examine the relationship between protein surface properties and selectivity in multimodal chromatographic systems. We hypothesized that multimodal ligands containing both hydrophobic and charged moieties would interact strongly with protein surface regions where charged groups and hydrophobic patches were in close spatial proximity. Protein surface property characterization tools were employed to identify the potential multimodal ligand binding regions on the Fab fragment of a humanized antibody and to evaluate the impact of mutations on surface charge and hydrophobicity. Twenty Fab variants were generated by site-directed mutagenesis, recombinant expression, and affinity purification. Column gradient experiments were carried out with the Fab variants in multimodal, cation-exchange, and hydrophobic interaction chromatographic systems. The results clearly indicated that selectivity in the multimodal system was different from the other chromatographic modes examined. Column retention data for the reduced charge Fab variants identified a binding site comprising light chain CDR1 as the main electrostatic interaction site for the multimodal and cation-exchange ligands. Furthermore, the multimodal ligand binding was enhanced by additional hydrophobic contributions as evident from the results obtained with hydrophobic Fab variants. The use of in silico protein surface property analyses combined with molecular biology techniques, protein expression, and chromatographic evaluations represents a previously undescribed and powerful approach for investigating multimodal selectivity with complex biomolecules.

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Section: Resultssupporting

confidence: 91%

Investigation of protein selectivity in multimodal chromatography using in silico designed Fab fragment variants

Karkov

Krogh

Woo

et al. 2015

Biotech & Bioengineering

Self Cite

View full text Add to dashboard Cite

show abstract

“…For example, a combination of nuclear magnetic resonance (NMR) spectroscopy, coarse-grained docking calculations and chromatographic experiments [23] revealed that the protein ubiquitin has a "preferred binding face" for interactions with the multimodal ligand Capto MMC. Molecular dynamics (MD) simulations of aqueous ubiquitin-ligand systems confirmed these results [24,25]. Other studies have focused on the role of arginine -another multimodal molecule -in eluting proteins from chromatographic columns.…”

Section: Introductionsupporting

confidence: 64%

Interactions of Multimodal Ligands with Proteins: Insights into Selectivity Using Molecular Dynamics Simulations

2015

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Fundamental understanding of protein-ligand interactions is important to the development of efficient bioseparations in multimodal chromatography. Here we employ molecular dynamics (MD) simulations to investigate the interactions of three different proteins--ubiquitin, cytochrome C, and α-chymotrypsinogen A, sampling a range of charge from +1e to +9e--with two multimodal chromatographic ligands containing similar chemical moieties--aromatic, carboxyl, and amide--in different structural arrangements. We use a spherical harmonic expansion to analyze ligand and individual moiety density profiles around the proteins. We find that the Capto MMC ligand, which contains an additional aliphatic group, displays stronger interactions than Nuvia CPrime ligand with all three proteins. Studying the ligand densities at the moiety level suggests that hydrophobic interactions play a major role in determining the locations of high ligand densities. Finally, the greater structural flexibility of the Capto MMC ligand compared to that of the Nuvia cPrime ligand allows for stronger structural complementarity and enables stronger hydrophobic interactions. These subtle and not-so-subtle differences in binding affinities and modalities for multimodal ligands can result in significantly different binding behavior towards proteins with important implications for bioprocessing.

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“…User-defined model(option_flag=1300) (9) In the above equation, ni * denotes the equilibrium concentration of solute in the pore of adsorbed particles, and Ci denotes the concentration of solute in the fluidized bed. Other symbols are model constants that are empirically derived or predicted.…”

Section: High Performance Adsorption Process Modelingmentioning

confidence: 99%

“…The derived values are generated and produced as 4 result files through the output file generator. Each created file is played back as a curved graph or visualized video at the final output stage [9].…”

Section: Simulation Platform System Designmentioning

confidence: 99%

Construction of Simulation for Adsorption Process Visualization

2019

IJRTE

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Recently, with the development of biotechnology, much interest has been focused on chromatographic adsorption processes for separation of biomolecules such as proteins, which are important. The efficiency of the adsorption process was improved by visualizing the data by simulation. The system visualized numerical results according to adsorption process modeling easily and efficiently. It is possible to predict the expected result even if the numerical data is inputted without going through the conventional experiment. The purification process of biological and chemical processes may involve several stages of chromatographic separation processes. The system visualizes the results of the simulation according to the adsorption process modeling or makes it appear as a curve graph. Therefore, in this paper, we design and implement a simulation system for adsorption process modeling. The currently developed simulation visualization system is a visualization of the result values that are driven by the existing engine, so future work will enable the engine to operate in the system itself

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Probing multimodal ligand binding regions on ubiquitin using nuclear magnetic resonance, chromatography, and molecular dynamics simulations

Cited by 30 publications

References 47 publications

Investigation of protein selectivity in multimodal chromatography using in silico designed Fab fragment variants

Investigation of protein selectivity in multimodal chromatography using in silico designed Fab fragment variants

Interactions of Multimodal Ligands with Proteins: Insights into Selectivity Using Molecular Dynamics Simulations

Construction of Simulation for Adsorption Process Visualization

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