{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,6]],"date-time":"2026-10-06T21:24:37Z","timestamp":1791321877404,"version":"4.3.1"},"reference-count":45,"publisher":"Wiley","issue":"9","license":[{"start":{"date-parts":[[2011,8,3]],"date-time":"2011-08-03T00:00:00Z","timestamp":1312329600000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/onlinelibrary.wiley.com\/termsAndConditions#vor"}],"funder":[{"name":"National Science Foundation","award":["MCB-0953323 (J.R.H.)"],"award-info":[{"award-number":["MCB-0953323 (J.R.H.)"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Protein Science"],"published-print":{"date-parts":[[2011,9]]},"abstract":"<jats:title>Abstract<\/jats:title><jats:p>There is growing interest in the development of protein switches, which are proteins whose function, such as binding a target molecule, can be modulated through environmental triggers. Efforts to engineer highly pH sensitive protein\u2013protein interactions typically rely on the rational introduction of ionizable groups in the protein interface. Such experiments are typically time intensive and often sacrifice the protein's affinity at the permissive pH. The underlying thermodynamics of proton\u2010linkage dictate that the presence of multiple ionizable groups, which undergo a p<jats:italic>K<\/jats:italic><jats:sub>a<\/jats:sub>change on protein binding, are necessary to result in highly pH\u2010dependent binding. To test this hypothesis, a novel combinatorial histidine library was developed where every possible combination of histidine and wild\u2010type residue is sampled throughout the interface of a model anti\u2010RNase A single domain VHH antibody. Antibodies were coselected for high\u2010affinity binding and pH\u2010sensitivity using an<jats:italic>in vitro<\/jats:italic>, dual\u2010function selection strategy. The resulting antibodies retained near wild\u2010type affinity yet became highly sensitive to small decreases in pH, drastically decreasing their binding affinity, due to the incorporation of multiple histidine groups. Several trends were observed, such as histidine \u201chot\u2010spots,\u201d which will help enhance the development of pH switch proteins as well as increase our understanding of the role of ionizable residues in protein interfaces. Overall, the combinatorial approach is rapid, general, and robust and should be capable of producing highly pH\u2010sensitive protein affinity reagents for a number of different applications.<\/jats:p>","DOI":"10.1002\/pro.696","type":"journal-article","created":{"date-parts":[[2011,7,15]],"date-time":"2011-07-15T14:05:34Z","timestamp":1310738734000},"page":"1619-1631","source":"Crossref","is-referenced-by-count":81,"title":["A combinatorial histidine scanning library approach to engineer highly pH\u2010dependent protein switches"],"prefix":"10.1002","volume":"20","author":[{"given":"Megan L.","family":"Murtaugh","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Sean W.","family":"Fanning","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Tressa M.","family":"Sharma","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Alexandra 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