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Structure, aggregation dynamics and crystallization of superfolder green fluorescence protein: Effect of long alkyl-chain imidazolium ionic liquids

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journal contribution
posted on 2023-11-17, 04:00 authored by Qi Han, Connie DarmaninConnie Darmanin, Carlos J Rosado, Nathalia Vieira Veríssimo, Jorge FB Pereira, Gary Bryant, Calum J Drummond, Tamar L Greaves
Green fluorescent protein (GFP) and its variants are widely used in medical and biological research, especially acting as indicators of protein structural integrity, protein-protein interactions and as biosensors. This study employs superfolder GFP (sfGFP) to investigate the impact of varying alkyl chain length of 1-Cn-3-methylimidazolium chloride ionic liquid (IL) series ([Cnmim]Cl, n = 2, 4, 6, 8, 10, 12) on the protein fluorescence, structure, hydration, aggregation dynamics and crystallization behaviour. The results revealed a concentration-dependent decrease in the sfGFP chromophore, particularly in long alkyl chain ILs ([C10mim]Cl and [C12mim]Cl). Tryptophan (Trp) fluorescence showed the quenching rate increased with longer alkyl chains indicating a nonpolar interaction between Trp57 and the alkyl chain. Secondary structural changes were observed at the high IL concentration of 1.5 M in [C10mim]Cl and [C12mim]Cl. Small-angle X-ray scattering (SAXS) indicated relatively stable protein sizes, but with IL aggregates present in [C10mim]Cl and [C12mim]Cl solutions. Dynamic light scattering (DLS) data showed increased protein size and aggregation with longer alkyl chain ILs. Notably, ILs and salts, excluding [C2mim]Cl, promoted sfGFP crystallization. This study emphasizes the influence of the cation alkyl chain length and concentration on protein stability and aggregation, providing insights into utilizing IL solvents for protein stabilization and crystallization purposes.


The authors acknowledge funding support from the Australian Research Council under its Discovery grant Scheme for this work (DP230101712). Jorge F. B. Pereira acknowledges the funding for CIEPQPF, which is supported by the Fundação para a Ciência e a Tecnologia (FCT) through the projects UIDB/EQU/00102/2020 and UIDP/EQU/00102/2020.


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International Journal of Biological Macromolecules




Part 7

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Rights Statement

© 2023 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY license (