{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,1]],"date-time":"2026-09-01T19:12:42Z","timestamp":1788289962786,"version":"build-2803163510"},"reference-count":68,"publisher":"American Chemical Society (ACS)","issue":"12","license":[{"start":{"date-parts":[[2015,6,3]],"date-time":"2015-06-03T00:00:00Z","timestamp":1433289600000},"content-version":"vor","delay-in-days":365,"URL":"https:\/\/pubs.acs.org\/page\/policy\/authorchoice_termsofuse.html"}],"funder":[{"DOI":"10.13039\/100000002","name":"National Institutes of Health","doi-asserted-by":"publisher","award":["SC3-GM089634"],"award-info":[{"award-number":["SC3-GM089634"]}],"id":[{"id":"10.13039\/100000002","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"published-print":{"date-parts":[[2014,6,16]]},"abstract":"<jats:title>Abstract<\/jats:title>\n                  <jats:p>Zinc finger proteins that bind Zn(II) using a Cys2His2 coordination motif within a \u03b2\u03b2\u03b1 protein fold are the most abundant DNA binding transcription factor domains in eukaryotic systems. These classic zinc fingers are typically unfolded in the apo state and spontaneously fold into their functional \u03b2\u03b2\u03b1 folds upon incorporation of Zn(II). These metal-induced protein folding events obscure the free energy cost of protein folding by coupling the protein folding and metal-ion binding thermodynamics. Herein, we determine the formation constant of a Cys2His2\/\u03b2\u03b2\u03b1 zinc finger domain, the C-terminal finger of the Wilms\u2019 tumor suppressor protein (WT1-4), for the purposes of determining its free energy cost of protein folding. Measurements of individual conditional dissociation constants, Kd values, at pH values from 5 to 9 were determined using fluorescence spectroscopy by direct or competition titration. Potentiometric titrations of apo-WT1-4 followed by NMR spectroscopy provided the intrinsic pKa values of the Cys2His2 residues, and corresponding potentiometric titrations of Zn(II)\u2013WT1-4 followed by fluorescence spectroscopy yielded the effective pKaeff values of the Cys2His2 ligands bound to Zn(II). The Kd, pKa, and pKaeff values were combined in a minimal, complete equilibrium model to yield the pH-independent formation constant value for Zn(II)\u2013WT1-4, KfML value of 7.5 \u00d7 1012 M\u20131, with a limiting Kd value of 133 fM. This shows that Zn(II) binding to the Cys2His2 site in WT1-4 provides at least \u221217.6 kcal\/mol in driving force to fold the protein scaffold. A comparison of the conditional dissociation constants of Zn(II)\u2013WT1-4 to those from the model peptide Zn(II)\u2013GGG\u2013Cys2His2 over the pH range 5.0 to 9.0 and a comparison of their pH-independent KfML values demonstrates that the free energy cost of protein folding in WT1-4 is less than +2.1 kcal\/mol. These results validate our GGG model system for determining the cost of protein folding in natural zinc finger proteins and support the conclusion that the cost of protein folding in most zinc finger proteins is \u2264+4.2 kcal\/mol, a value that pales in comparison to the free energy contribution of Zn(II) binding, \u221217.6 kcal\/mol.<\/jats:p>","DOI":"10.1021\/ic500862b","type":"journal-article","created":{"date-parts":[[2014,6,3]],"date-time":"2014-06-03T14:27:12Z","timestamp":1401805632000},"page":"6309-6320","source":"Crossref","is-referenced-by-count":26,"title":["Characterization\nof the Zn(II) Binding Properties\nof the Human Wilms\u2019 Tumor Suppressor Protein C-terminal\nZinc Finger Peptide"],"prefix":"10.1021","volume":"53","author":[{"given":"Ka Lam","family":"Chan","sequence":"first","affiliation":[{"name":"Brooklyn College , , 2900 Bedford Avenue , , ,","place":["Brooklyn, New York, United States, 11210"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Inna","family":"Bakman","sequence":"additional","affiliation":[{"name":"Brooklyn College , , 2900 Bedford Avenue , , ,","place":["Brooklyn, New York, United States, 11210"]},{"name":"The City University of New York , , , ,","place":["New York, New York, United States, 10016"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Amy R.","family":"Marts","sequence":"additional","affiliation":[{"name":"Miami University , , , ,","place":["Oxford, Ohio, United States, 45056"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yuksel","family":"Batir","sequence":"additional","affiliation":[{"name":"Brooklyn College , , 2900 Bedford Avenue , , ,","place":["Brooklyn, New York, United States, 11210"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Terry L.","family":"Dowd","sequence":"additional","affiliation":[{"name":"Brooklyn College , , 2900 Bedford Avenue , , ,","place":["Brooklyn, New York, United States, 11210"]},{"name":"The City University of New York , , , ,","place":["New York, New York, United States, 10016"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"David L.","family":"Tierney","sequence":"additional","affiliation":[{"name":"Miami University , , , ,","place":["Oxford, Ohio, United States, 45056"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Brian R.","family":"Gibney","sequence":"additional","affiliation":[{"name":"Brooklyn College , , 2900 Bedford Avenue , , ,","place":["Brooklyn, New York, United States, 11210"]},{"name":"The City University of New York , , , ,","place":["New York, New York, United States, 10016"]}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"316","published-online":{"date-parts":[[2014,6,3]]},"reference":[{"key":"2026090114125482600_cit1","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1017\/S0033583510000089","volume":"43","author":"Klug","year":"2010","journal-title":"Q. 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