Engineering fungal antifreeze proteins through ‘EKylation’ and mechanism analysis

doi:10.12211/2096-8280.2025-019

Abstract

Abstract:

Antifreeze proteins (AFPs), a functionally unique and diverse class of biomolecules, possess the ability to depress the freezing point of aqueous solutions non-colligatively and inhibit the damaging process of ice recrystallization. These critical activities stem from their surface-adsorption mechanism at the ice-water interface, preventing ice crystal growth and maturation. Consequently, the strategic development of novel AFP analogues exhibiting significantly enhanced activity and stability represents an area of substantial research interest and practical value. Herein, we introduce and implement an "EKylation" strategy designed to rationally engineer the AFP (RCSB ID:5B5H) derived from the freeze-tolerant fungus Typhula ishikariensis. This approach specifically leverages the charge-tunable properties inherent to zwitterionic peptides to modify protein surfaces. We chemically coupled the synthetic zwitterionic polypeptide (EK)₂₀, comprising alternating glutamate (E, negatively charged) and lysine (K, positively charged) residues, to the N-terminal structural domain of the wild-type 5B5H, which yielded the recombinant conjugate, designated 5B5H-EK. Comprehensive biophysical characterization revealed that the 5B5H-EK conjugate exhibited markedly enhanced structural stability compared to its unmodified counterpart. Crucially, this conjugation strategy led to a significant functional improvement, with the modified protein demonstrating a thermal hysteresis (TH) activity of 27.8%, representing a substantial enhancement over the wild-type AFP. Further insight into the molecular basis of this activity boost was gained through molecular dynamics (MD) simulations. These simulations indicated that the core ice crystal binding surface architecture of 5B5H-EK was largely preserved. Therefore, the conjugated zwitterionic chain significantly augmented the protein's inherent ability to inhibit ice crystal growth and bind tenaciously to the ice surface. Analysis of water dynamics near the modified protein surface suggested that the (EK)₂₀ chain promotes the formation of an extensive, short-range ordered hydration shell, effectively structuring interfacial water molecules into an "ice-water-like" layer distinct from bulk water. This engineered interfacial hydration likely contributes synergistically to the enhanced TH performance by reinforcing the AFP's anchoring to the quasi-liquid layer and facilitating greater surface coverage. In conclusion, this study proposes a novel and effective protein engineering strategy, utilizing zwitterionic peptide conjugation ("EKylation"), for generating highly efficient antifreeze proteins and informs the rational design of next-generation, environmentally adaptable antifreeze materials.

Key words: EKylation, antifreeze proteins, zwitterionic peptides, thermal hysteresis activity, molecular dynamics modelling

摘要：

抗冻蛋白（Antifreeze proteins， AFPs）是一类通过非依数性机制降低冰点并有效抑制冰晶生长的生物大分子，广泛分布于极地鱼类、昆虫及耐寒微生物等生物体内以维持其低温适应性，在食品冷冻保存、低温医学及低温生物技术等领域具有广泛应用，开发高活性AFPs具有重要研究价值。本研究利用“EKylation”策略对雪腐病核瑚菌（Typhula ishikariensis）来源的AFPs（RCSB ID：5B5H）开展分子改造研究。基于两性离子多肽的电荷可调特性与蛋白质稳定化机制，将两性离子多肽（EK）₂₀定点偶联至5B5H的N端结构域，经两性离子改造的重组蛋白5B5H-EK高级结构保持稳定，热滞活性较野生型提升27.8%。分子动力学模拟进一步揭示5B5H-EK的冰晶结合面并未发生改变，且具有更强的抑制冰晶生长能力和冰晶结合能力，促使水分子形成短程有序的"类冰水"结构。该研究为理性设计高效抗冻蛋白及环境适应性抗冻材料提供了新策略。

关键词: EKylation, 抗冻蛋白, 两性离子多肽, 热滞活性, 分子动力学模拟

CLC Number:

CUI Zhongxin, WANG Yi, ZHANG Lei, QI Haishan. Engineering fungal antifreeze proteins through ‘EKylation’ and mechanism analysis[J]. Synthetic Biology Journal, DOI: 10.12211/2096-8280.2025-019.

崔忠信, 王怡, 张雷, 齐海山. “EKylation”策略改造真菌抗冻蛋白及机制解析[J]. 合成生物学, DOI: 10.12211/2096-8280.2025-019.

Figures/Tables 6

Table 1 Primer sequence

蛋白名称	5′端引物序列	3′端引物序列
5B5H	TGGGCAGCAGCCATCATCAT	TGGTGGTGGTGGTGCTC
5B5H-EK	GTGGGCAGCAGCCATCATCAT	TGGTGGTGGTGGTGCTCT

Table 2 the amino acid sequence of 5B5H and 5B5H-EK

种类	序列
5B5H	AGPTAVPLGTAGNYAILASAGVSTVPQSVITGAVGLSPAAATFLTGFSLTMSSTGTFSTSTQVTGQLTAADYGTPTPSILTTAIGDMGTAYVNAATRSGPNFLEIYTGALGGKILPPGLYKWTSPVGASADFTIIGTSTDTWIFQIAGTLGLAAGKKIILAGGAQAKNIVWVVAGAVSIEAGAKFEGVILAKTAVTLKTGSSLNGRILSQTAVALQKATVVQK
5B5H-EK	EKEKEKEKEKEKEKEKEKEKEKEKEKEKEKEKEKEKEKEKAGPTAVPLGTAGNYAILASAGVSTVPQSVITGAVGLSPAAATFLTGFSLTMSSTGTFSTSTQVTGQLTAADYGTPTPSILTTAIGDMGTAYVNAATRSGPNFLEIYTGALGGKILPPGLYKWTSPVGASADFTIIGTSTDTWIFQIAGTLGLAAGKKIILAGGAQAKNIVWVVAGAVSIEAGAKFEGVILAKTAVTLKTGSSLNGRILSQTAVALQKATVVQK

Fig. 1 Conformation resolution of EK, 5B5H and 5B5H-EK(a) 5B5H modification site exploration (b) 5B5H (N-terminal modification) spatial structure (c) 5B5H (C-terminal modification) spatial structure (d) 5B5H aqueous environment Rg simulation results (e) 5B5H (N-terminal modification) aqueous environment Rg simulation results (f) 5B5H (C-terminal modification) aqueous environment Rg simulation results (g) 5B5H aqueous environment RMSD simulation results (h) 5B5H (N-terminal modification) aqueous environment RMSD simulation results (i) 5B5H (C-terminal modification) aqueous environment RMSD simulation results

Fig. 2 Fermentation, purification and heat-lag activity measurements of 5B5H and 5B5H-EK(a) Plasmid of 5B5H-EK (b) Purification results of 5B5H and 5B5H-EK proteins (c) DSC results of 5B5H and 5B5H-EK at 2 mg/mL (d) Thermal hysteresis activities of 5B5H and 5B5H-EK at different concentrations

Fig. 3 5B5H and 5B5H-EK ice-water environment simulation results(a) Ice-water environment inhibition of ice crystal growth results of EK,5B5H and 5B5H-EK (b) F3 of 5B5H and 5B5H-EK in entire ice environment (c) F4 of 5B5H and 5B5H-EK in entire ice environment

Fig. 4 5B5H and 5B5H-EK ice-crystal bonding surface resolution(a) Conformation of 5B5H's IBF (b) Conformation of 5B5H-EK's IBF (c) 5B5H ice-crystal bonding surface stability (d) 5B5H-EK ice-crystal bonding surface stability (e) 5B5H and 5B5H-EK with ice-crystal bonding energies (f) Hydrodynamics around the ice-crystal bonding surface of 5B5H and 5B5H-EK

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