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

Qi Han; Connie Darmanin; Carlos J Rosado; Nathalia Vieira Veríssimo; Jorge F B Pereira; Gary Bryant; Calum J Drummond; Tamar L Greaves

doi:10.1016/j.ijbiomac.2023.127456

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

Int J Biol Macromol. 2023 Dec 31;253(Pt 7):127456. doi: 10.1016/j.ijbiomac.2023.127456. Epub 2023 Oct 14.

Authors

Qi Han¹, Connie Darmanin², Carlos J Rosado³, Nathalia Vieira Veríssimo⁴, Jorge F B Pereira⁵, Gary Bryant⁶, Calum J Drummond⁶, Tamar L Greaves⁷

Affiliations

¹ School of Science, STEM College, RMIT University, Melbourne, VIC 3000, Australia. Electronic address: qi.han@rmit.edu.au.
² La Trobe Institute for Molecular Science, Department of Mathematical and Physical Sciences, School of Computing Engineering and Mathematical Science, La Trobe University, Bundoora, VIC 3086, Australia.
³ Department of Diabetes, Central Clinical School, Monash University, VIC 3004, Australia; Department of Biochemistry, Monash University, VIC 3800, Australia.
⁴ School of Pharmaceutical Sciences, São Paulo University (USP), Av. Prof. Lineu Prestes, no. 580, B16, 05508-000, Cidade de Universitária, São Paulo, SP, Brazil.
⁵ University of Coimbra, CIEPQPF, Department of Chemical Engineering, Rua Sílvio Lima, Pólo II - Pinhal de Marrocos, 3030-790 Coimbra, Portugal.
⁶ School of Science, STEM College, RMIT University, Melbourne, VIC 3000, Australia.
⁷ School of Science, STEM College, RMIT University, Melbourne, VIC 3000, Australia. Electronic address: tamar.greaves@rmit.edu.au.

PMID: 37844813
DOI: 10.1016/j.ijbiomac.2023.127456

Abstract

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-C_n-3-methylimidazolium chloride ionic liquid (IL) series ([C_nmim]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 fluorescence, 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.

Keywords: Dynamic light scattering; Green fluorescent protein; Imidazolium chloride; Ionic liquids; Protein crystallization; Small-angle X-ray scattering (SAXS).

MeSH terms

Crystallization
Green Fluorescent Proteins / genetics
Ionic Liquids* / chemistry
Scattering, Small Angle
X-Ray Diffraction

Substances

Green Fluorescent Proteins
Ionic Liquids