{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,3,14]],"date-time":"2026-03-14T01:47:05Z","timestamp":1773452825155,"version":"3.50.1"},"reference-count":35,"publisher":"MDPI AG","issue":"18","license":[{"start":{"date-parts":[[2023,9,20]],"date-time":"2023-09-20T00:00:00Z","timestamp":1695168000000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"National Eye Institute","award":["ZIA EY000281-30"],"award-info":[{"award-number":["ZIA EY000281-30"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["IJMS"],"abstract":"<jats:p>Aromatic residues forming tyrosine corners within Greek key motifs are critical for the folding, stability, and order of \u03b2\u03b3-crystallins and thus lens transparency. To delineate how a double amino acid substitution in an N-terminal-domain tyrosine corner of the CRYGS mutant p.F10_Y11delinsLN causes juvenile autosomal dominant cortical lamellar cataracts, human \u03b3S-crystallin c-DNA was cloned into pET-20b (+) and a p.F10_Y11delinsLN mutant was generated via site-directed mutagenesis, overexpressed, and purified using ion-exchange and size-exclusion chromatography. Structure, stability, and aggregation properties in solution under thermal and chemical stress were determined using spectrofluorimetry and circular dichroism. In benign conditions, the p.F10_Y11delinsLN mutation does not affect the protein backbone but alters its tryptophan microenvironment slightly. The mutant is less stable to thermal and GuHCl-induced stress, undergoing a two-state transition with a midpoint of 60.4 \u00b0C (wild type 73.1 \u00b0C) under thermal stress and exhibiting a three-state transition with midpoints of 1.25 and 2.59 M GuHCl (wild type: two-state transition with Cm = 2.72 M GuHCl). The mutant self-aggregates upon heating at 60 \u00b0C, which is inhibited by \u03b1-crystallin and reducing agents. Thus, the F10_Y11delinsLN mutation in human \u03b3S-crystallin impairs the protein\u2019s tryptophan microenvironment, weakening its stability under thermal and chemical stress, resulting in self-aggregation, lens opacification, and cataract.<\/jats:p>","DOI":"10.3390\/ijms241814332","type":"journal-article","created":{"date-parts":[[2023,9,20]],"date-time":"2023-09-20T21:26:32Z","timestamp":1695245192000},"page":"14332","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":3,"title":["Human \u03b3S-Crystallin Mutation F10_Y11delinsLN in the First Greek Key Pair Destabilizes and Impairs Tight Packing Causing Cortical Lamellar Cataract"],"prefix":"10.3390","volume":"24","author":[{"ORCID":"https:\/\/orcid.org\/0000-0003-0223-9464","authenticated-orcid":false,"given":"Venkata Pulla Rao","family":"Vendra","sequence":"first","affiliation":[{"name":"Ophthalmic Molecular Genetics Section, Ophthalmic Genetics and Visual Function Branch, National Eye Institute, National Institutes of Health, Bethesda, MD 20892, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Christian","family":"Ostrowski","sequence":"additional","affiliation":[{"name":"Ophthalmic Molecular Genetics Section, Ophthalmic Genetics and Visual Function Branch, National Eye Institute, National Institutes of Health, Bethesda, MD 20892, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Marzena A.","family":"Dyba","sequence":"additional","affiliation":[{"name":"Biophysics Resource in the Center for Structural Biology, National Cancer Institute, National Institutes of Health, Frederick, MD 21702, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-7134-0374","authenticated-orcid":false,"given":"Sergey G.","family":"Tarasov","sequence":"additional","affiliation":[{"name":"Biophysics Resource in the Center for Structural Biology, National Cancer Institute, National Institutes of Health, Frederick, MD 21702, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-4788-0362","authenticated-orcid":false,"given":"J. Fielding","family":"Hejtmancik","sequence":"additional","affiliation":[{"name":"Ophthalmic Molecular Genetics Section, Ophthalmic Genetics and Visual Function Branch, National Eye Institute, National Institutes of Health, Bethesda, MD 20892, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1968","published-online":{"date-parts":[[2023,9,20]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"32","DOI":"10.1159\/000267371","article-title":"Refractive index of the human lens surface measured with an optic fibre sensor","volume":"26","author":"Pierscionek","year":"1994","journal-title":"Ophthalmic Res."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"415","DOI":"10.1038\/302415a0","article-title":"Short-range order of crystallin proteins accounts for eye lens transparency","volume":"302","author":"Delaye","year":"1983","journal-title":"Nature"},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"459","DOI":"10.1364\/AO.10.000459","article-title":"Theory of transparency of the eye","volume":"10","author":"Benedek","year":"1971","journal-title":"Appl. Opt."},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"10449","DOI":"10.1073\/pnas.89.21.10449","article-title":"Alpha-crystallin can function as a molecular chaperone","volume":"89","author":"Horwitz","year":"1992","journal-title":"Proc. Nat. Acad. Sci. USA"},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"26","DOI":"10.1186\/1479-7364-6-26","article-title":"The human crystallin gene families","volume":"6","author":"Wistow","year":"2012","journal-title":"Hum. Genom."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"1927","DOI":"10.1002\/pro.5560031104","article-title":"The tyrosine corner: A feature of most Greek key beta-barrel proteins","volume":"3","author":"Hemmingsen","year":"1994","journal-title":"Protein Sci."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"333","DOI":"10.1016\/j.bbagen.2015.06.007","article-title":"Gamma crystallins of the human eye lens","volume":"1860 Pt B","author":"Vendra","year":"2016","journal-title":"Biochim. Biophys. Acta"},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"23","DOI":"10.1126\/science.3892686","article-title":"Aromatic-aromatic interaction: A mechanism of protein structure stabilization","volume":"229","author":"Burley","year":"1985","journal-title":"Science"},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"15458","DOI":"10.1074\/jbc.273.25.15458","article-title":"pi-Stacking interactions. Alive and well in proteins","volume":"273","author":"McGaughey","year":"1998","journal-title":"J. Biol. Chem."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"3257","DOI":"10.1534\/g3.117.300109","article-title":"High-Throughput Genetic Screening of 51 Pediatric Cataract Genes Identifies Causative Mutations in Inherited Pediatric Cataract in South Eastern Australia","volume":"7","author":"Javadiyan","year":"2017","journal-title":"G3"},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"1864","DOI":"10.1021\/acs.biochem.2c00628","article-title":"The Y46D Mutation Destabilizes Dense Packing of the Second Greek Key Pair of Human gammaC-Crystallin Causing Congenital Nuclear Cataracts","volume":"62","author":"Vendra","year":"2023","journal-title":"Biochemistry"},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"365","DOI":"10.1016\/S0076-6879(98)90032-5","article-title":"Lens alpha-crystallin: Chaperone-like properties","volume":"290","author":"Horwitz","year":"1998","journal-title":"Methods Enzymol."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"7334","DOI":"10.1021\/bi900467a","article-title":"The G18V CRYGS Mutation Associated with Human Cataracts Increases \u03b3S-Crystallin Sensitivity to Thermal and Chemical Stress","volume":"48","author":"Ma","year":"2009","journal-title":"Biochemistry"},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"3915","DOI":"10.1021\/bi00838a006","article-title":"Dimer formation from 1-amino-8-naphthalenesulfonate catalyzed by bovine serum albumin. A new fluorescent molecule with exceptional binding properties","volume":"8","author":"Rosen","year":"1969","journal-title":"Biochemistry"},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"181","DOI":"10.1007\/s10895-007-0156-6","article-title":"Sensitive spectroscopic detection of large and denatured protein aggregates in solution by use of the fluorescent dye Nile red","volume":"17","author":"Sutter","year":"2007","journal-title":"J. Fluoresc."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"6220","DOI":"10.1074\/jbc.272.10.6220","article-title":"Conformational and functional differences between recombinant human lens alphaA- and alphaB-crystallin","volume":"272","author":"Sun","year":"1997","journal-title":"J. Biol.Chem."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"10705","DOI":"10.1021\/bi800499k","article-title":"Mechanism of the efficient tryptophan fluorescence quenching in human gammaD-crystallin studied by time-resolved fluorescence","volume":"47","author":"Chen","year":"2008","journal-title":"Biochemistry"},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"641","DOI":"10.1016\/0022-2836(87)90038-6","article-title":"Interior and surface of monomeric proteins","volume":"196","author":"Miller","year":"1987","journal-title":"J. Mol. Biol."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"325","DOI":"10.1016\/j.bbagen.2015.09.022","article-title":"Increased hydrophobic surface exposure in the cataract-related G18V variant of human gammaS-crystallin","volume":"1860 Pt B","author":"Khago","year":"2016","journal-title":"Biochim. Biophys. Acta"},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"108662","DOI":"10.1016\/j.exer.2021.108662","article-title":"Inherited cataracts: Genetic mechanisms and pathways new and old","volume":"209","author":"Shiels","year":"2021","journal-title":"Exp. Eye Res."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"8","DOI":"10.1167\/iovs.63.4.8","article-title":"Patterns of Crystallin Gene Expression in Differentiation State Specific Regions of the Embryonic Chicken Lens","volume":"63","author":"Ma","year":"2022","journal-title":"Investig. Ophthalmol. Vis. Sci."},{"key":"ref_22","doi-asserted-by":"crossref","unstructured":"Kraus, C.L. (2020). Pediatric Cataract Surgery and IOL Implantation, Springer.","DOI":"10.1007\/978-3-030-38938-3"},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"513","DOI":"10.1002\/pro.583","article-title":"Contributions of aromatic pairs to the folding and stability of long-lived human gammaD-crystallin","volume":"20","author":"Kong","year":"2011","journal-title":"Protein Sci."},{"key":"ref_24","doi-asserted-by":"crossref","unstructured":"Moreau, K.L., and King, J.A. (2012). Cataract-causing defect of a mutant gamma-crystallin proceeds through an aggregation pathway which bypasses recognition by the alpha-crystallin chaperone. PLoS ONE, 7.","DOI":"10.1371\/journal.pone.0037256"},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"917","DOI":"10.1006\/bbrc.1998.9242","article-title":"The chaperone-like alpha-crystallin forms a complex only with the aggregation-prone molten globule state of alpha-lactalbumin","volume":"249","author":"Rajaraman","year":"1998","journal-title":"Biochem. Biophys. Res. Commun."},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"2221","DOI":"10.1016\/j.str.2013.09.017","article-title":"Preferential and specific binding of human alphaB-crystallin to a cataract-related variant of gammaS-crystallin","volume":"21","author":"Kingsley","year":"2013","journal-title":"Structure"},{"key":"ref_27","first-page":"1231","article-title":"Structural analysis of the mutant protein D26G of human gammaS-crystallin, associated with Coppock cataract","volume":"19","author":"Karri","year":"2013","journal-title":"Mol. Vis."},{"key":"ref_28","first-page":"771","article-title":"Structural study of the G57W mutant of human gamma-S-crystallin, associated with congenital cataract","volume":"22","author":"Khan","year":"2016","journal-title":"Mol. Vis."},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"498","DOI":"10.1016\/j.bpj.2010.12.3691","article-title":"Separating instability from aggregation propensity in gammaS-crystallin variants","volume":"100","author":"Brubaker","year":"2011","journal-title":"Biophys. J."},{"key":"ref_30","doi-asserted-by":"crossref","unstructured":"Vendra, V.P., Chandani, S., and Balasubramanian, D. (2012). The mutation V42M distorts the compact packing of the human gamma-S-crystallin molecule, resulting in congenital cataract. PLoS ONE, 7.","DOI":"10.1371\/journal.pone.0051401"},{"key":"ref_31","unstructured":"Fasman, G.D. (1968). Handbook of Biochemistry, CRC Press."},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"83","DOI":"10.1016\/0003-2697(92)90046-A","article-title":"Analysis of the circular dichroism spectrum of proteins using the convex constraint algorithm: A practical guide","volume":"203","author":"Perczel","year":"1992","journal-title":"Anal. Biochem."},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"266","DOI":"10.1016\/0076-6879(86)31045-0","article-title":"Determination and analysis of urea and guanidine hydrochloride denaturation curves","volume":"131","author":"Pace","year":"1986","journal-title":"Methods Enzymol."},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"257","DOI":"10.1146\/annurev-physchem-032210-103531","article-title":"Role of solvation effects in protein denaturation: From thermodynamics to single molecules and back","volume":"62","author":"England","year":"2011","journal-title":"Annu. Rev. Phys. Chem."},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"763","DOI":"10.1016\/j.str.2022.03.002","article-title":"Deamidation of the human eye lens protein gammaS-crystallin accelerates oxidative aging","volume":"30","author":"Mehrabi","year":"2022","journal-title":"Structure"}],"container-title":["International Journal of Molecular Sciences"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1422-0067\/24\/18\/14332\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,10]],"date-time":"2025-10-10T20:53:38Z","timestamp":1760129618000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1422-0067\/24\/18\/14332"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2023,9,20]]},"references-count":35,"journal-issue":{"issue":"18","published-online":{"date-parts":[[2023,9]]}},"alternative-id":["ijms241814332"],"URL":"https:\/\/doi.org\/10.3390\/ijms241814332","relation":{},"ISSN":["1422-0067"],"issn-type":[{"value":"1422-0067","type":"electronic"}],"subject":[],"published":{"date-parts":[[2023,9,20]]}}}