Preferential exclusion of sucrose from recombinant interleukin-1 receptor antagonist: Role in restricted conformational mobility and compaction of native state

Understanding the mechanism for sucrose-induced protein stabilization is important in many diverse fields, ranging from biochemistry and environmental physiology to pharmaceutical science. Timasheff and Lee [Lee, J. C. & Timasheff, S. N. (1981) J. Biol. Chem. 256, 7193–7201] have established that thermodynamic stabilization of proteins by sucrose is due to preferential exclusion of the sugar from the protein’s surface, which increases protein chemical potential. The current study measures the preferential exclusion of 1 M sucrose from a protein drug, recombinant interleukin 1 receptor antagonist (rhIL-1ra). It is proposed that the degree of preferential exclusion and increase in chemical potential are directly proportional to the protein surface area and that, hence, the system will favor the protein state with the smallest surface area. This mechanism explains the observed sucrose-induced restriction of rhIL-1ra conformational fluctuations, which were studied by hydrogen–deuterium exchange and cysteine reactivity measurements. Furthermore, infrared spectroscopy of rhlL-1ra suggested that a more ordered native conformation is induced by sucrose. Electron paramagnetic resonance spectroscopy demonstrated that in the presence of sucrose, spin-labeled cysteine 116 becomes more buried in the protein’s interior and that the hydrodynamic diameter of the protein is reduced. The preferential exclusion of sucrose from the protein and the resulting shift in the equilibrium between protein states toward the most compact conformation account for sucrose-induced effects on rhIL-1ra.

解析蔗糖诱导蛋白质稳定的作用机制，在生物化学、环境生理学乃至药物科学等诸多领域均具有重要研究价值。Timasheff与Lee[Lee, J. C. & Timasheff, S. N. (1981) J. Biol. Chem. 256, 7193–7201]已证实，蔗糖对蛋白质的热力学稳定作用源于其从蛋白质表面优先被排除，进而提升了蛋白质的化学势。本研究针对重组白细胞介素1受体拮抗剂（recombinant interleukin 1 receptor antagonist, rhIL-1ra）这一蛋白药物，测定了1 M蔗糖从其表面发生优先排除的具体情况。研究提出，优先排除程度与化学势提升幅度均与蛋白质表面积呈正比关系，因此体系会倾向于选择表面积最小的蛋白质构象。该机制可解释此前观测到的蔗糖诱导rhIL-1ra构象波动受限现象，该现象通过氢氘交换与半胱氨酸反应性测定得以研究。此外，针对rhIL-1ra的红外光谱分析表明，蔗糖可诱导形成更有序的天然构象。电子顺磁共振波谱分析证实，在蔗糖存在的条件下，被自旋标记的半胱氨酸116会更深地埋藏于蛋白质内部，且蛋白质的流体力学直径有所减小。蔗糖从蛋白质表面的优先排除，以及由此引发的蛋白质构象平衡向最紧凑构象方向的偏移，共同阐明了蔗糖对rhIL-1ra产生的各项作用效果。