Ionic liquids offer exciting possibilities for biocatalysis as solvent properties provide rare opportunities for customizable, energy-efficient bioprocessing. Unfortunately, proteins and enzymes are generally unstable in ionic liquids and several attempts have been made to explain why; however, a comprehensive understanding of the ionic liquid–protein interactions remains elusive. Here, we present an analytical framework (circular dichroism (CD), fluorescence, ultraviolet-visible (UV/Vis) and nuclear magnetic resonance (NMR) spectroscopies, and small-angle X-ray scattering (SAXS)) to probe the interactions, structure, and stability of a model protein (green fluorescent protein (GFP)) in a range (acetate, chloride, triflate) of pyrrolidinium and imidazolium salts. We demonstrate that measuring protein stability requires a similar holistic analytical framework, as opposed to single-technique assessments that provide misleading conclusions. We reveal information on site-specific ionic liquid–protein interactions, revealing that triflate (the least interacting anion) induces a contraction in the protein size that reduces the barrier to unfolding. Robust frameworks such as this are critical to advancing non-aqueous biocatalysis and avoiding pitfalls associated with single-technique investigations.

离子液体为生物催化提供了令人兴奋的可能性，因为溶剂特性为可定制、节能的生物加工提供了难得的机会。不幸的是，蛋白质和酶在离子液体中通常不稳定，并且已经进行了多次尝试来解释原因；然而，对离子液体-蛋白质相互作用的全面了解仍然难以捉摸。在这里，我们提出了一个分析框架（圆二色性 (CD)、荧光、紫外-可见 (UV/Vis) 和核磁共振 (NMR) 光谱以及小角度 X 射线散射 (SAXS)）来探测相互作用，模型蛋白（绿色荧光蛋白 (GFP)）在一系列吡咯烷盐和咪唑盐（乙酸盐、氯化物、三氟甲磺酸盐）中的结构和稳定性。我们证明测量蛋白质稳定性需要类似的整体分析框架，而不是提供误导性结论的单一技术评估。我们揭示了有关位点特异性离子液体-蛋白质相互作用的信息，表明三氟甲磺酸盐（相互作用最少的阴离子）会导致蛋白质大小收缩，从而降低展开的障碍。诸如此类的稳健框架对于推进非水生物催化和避免与单一技术研究相关的陷阱至关重要。