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However, the rational design of AFPTs is hindered by the unclear functional roles of non\u2010ice\u2010binding sites (NIBSs), especially whether the charge\u2010specific effect on ice inhibition exists. Here, we design a series of charged AFPTs and clarify the hidden contributions of the \u201cdark matter\u201d NIBSs, revealing their essential charge impact on the cryoprotective performance. Moderate net negative charges of NIBSs create a hydration\u2010mediated interfacial environment that facilitates AFPT adsorption onto ice crystals and enhances ice growth inhibition. However, excessive negative charges induce overly strong hydration layers that hinder effective interfacial contact and reduce solution pH, thereby leading to cytotoxicity. Positive charges promote ordered water arrangements conducive to ice growth. The optimal charged AFPT (a net charge of \u20102) exhibits a 3.5\u2010fold ice growth inhibition rate compared with other AFPTs, as well as possessing biocompatibility, thus enhancing the post\u2010thaw red blood cells recovery rate to above 93%. This work offers new insights on elucidating the structure\u2010activity relationship of AFPTs and provides a promising approach for developing a next\u2010generation cryoprotectant in clinical applications.<\/jats:p>","DOI":"10.1002\/smll.202511772","type":"journal-article","created":{"date-parts":[[2026,3,9]],"date-time":"2026-03-09T03:10:05Z","timestamp":1773025805000},"update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":2,"title":["Charge\u2010Tuning Ice Inhibition in Antifreeze Peptides"],"prefix":"10.1002","volume":"22","author":[{"given":"Yunqing","family":"Tian","sequence":"first","affiliation":[{"name":"Department of Bioengineering School of Synthetic Biology and Biomanufacturing State Key Laboratory of Synthetic Biology Frontier Science Center for Synthetic Biology Tianjin University  Tianjin 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