{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2025,11,2]],"date-time":"2025-11-02T16:40:15Z","timestamp":1762101615647,"version":"build-2065373602"},"reference-count":57,"publisher":"MDPI AG","issue":"4","license":[{"start":{"date-parts":[[2014,10,20]],"date-time":"2014-10-20T00:00:00Z","timestamp":1413763200000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Biomolecules"],"abstract":"<jats:p>Numerous human diseases are caused by protein folding defects where the  protein may become more susceptible to degradation or aggregation. Aberrant protein folding can affect the kinetic stability of the proteins even if these proteins appear to  be soluble in vivo. Experimental discrimination between functional properly folded and misfolded nonfunctional conformers is not always straightforward at near physiological conditions. The differences in the kinetic behavior of two initially folded frataxin clinical variants were examined using a high affinity chaperonin kinetic trap approach at 25 \u00b0C. The kinetically stable wild type frataxin (FXN) shows no visible partitioning onto  the chaperonin. In contrast, the clinical variants FXN-p.Asp122Tyr and FXN-p.Ile154Phe kinetically populate partial folded forms that tightly bind the GroEL chaperonin platform. The initially soluble FXN-p.Ile154Phe variant partitions onto GroEL more rapidly and  is more kinetically liable. These differences in kinetic stability were confirmed using differential scanning fluorimetry. The kinetic and aggregation stability differences of these variants may lead to the distinct functional impairments described in Friedreich\u2019s ataxia,  the neurodegenerative disease associated to frataxin functional deficiency. This chaperonin platform approach may be useful for identifying small molecule stabilizers since stabilizing ligands to frataxin variants should lead to a concomitant decrease in chaperonin binding.<\/jats:p>","DOI":"10.3390\/biom4040956","type":"journal-article","created":{"date-parts":[[2014,10,20]],"date-time":"2014-10-20T10:14:47Z","timestamp":1413800087000},"page":"956-979","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":4,"title":["Probing the Kinetic Stabilities of Friedreich\u2019s Ataxia Clinical Variants Using a Solid Phase GroEL Chaperonin  Capture Platform"],"prefix":"10.3390","volume":"4","author":[{"given":"Ana","family":"Correia","sequence":"first","affiliation":[{"name":"Instituto de Tecnologia Qu\u00edmica e Biol\u00f3gica Ant\u00f3nio Xavier, Universidade Nova de Lisboa,  Av. da Rep\u00fablica, EAN, Oeiras 2784-505, Portugal"}]},{"given":"Subhashchandra","family":"Naik","sequence":"additional","affiliation":[{"name":"Department of Biochemistry and Molecular Biology, Hemenway Life Sciences Innovation Center (HLSIC), University of Kansas Medical Center, 3901 Rainbow Blvd., Kansas City, KS 66160, USA"}]},{"given":"Mark","family":"Fisher","sequence":"additional","affiliation":[{"name":"Department of Biochemistry and Molecular Biology, Hemenway Life Sciences Innovation Center (HLSIC), University of Kansas Medical Center, 3901 Rainbow Blvd., Kansas City, KS 66160, USA"}]},{"given":"Cl\u00e1udio","family":"Gomes","sequence":"additional","affiliation":[{"name":"Instituto de Tecnologia Qu\u00edmica e Biol\u00f3gica Ant\u00f3nio Xavier, Universidade Nova de Lisboa,  Av. da Rep\u00fablica, EAN, Oeiras 2784-505, Portugal"}]}],"member":"1968","published-online":{"date-parts":[[2014,10,20]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"4449","DOI":"10.1073\/pnas.1323268111","article-title":"Small molecule probes to quantify the functional fraction of a specific protein in a cell with minimal folding equilibrium shifts","volume":"111","author":"Liu","year":"2014","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"19128","DOI":"10.3390\/ijms140919128","article-title":"Small molecules present in the cerebrospinal fluid metabolome influence superoxide dismutase 1 aggregation","volume":"14","author":"Cristovao","year":"2013","journal-title":"Int. J. Mol. Sci."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"57","DOI":"10.1007\/s10969-008-9053-8","article-title":"Strategies for folding of affinity tagged proteins using GroEL and osmolytes","volume":"10","author":"Katayama","year":"2009","journal-title":"J. Struct. Funct. Genomics"},{"key":"ref_4","unstructured":"Marques, A.R., Tost\u00f5es, R., Alves, E., Gomes, C.M., and Martinho, R.G. (2008, January 22\u201326). Use of drosophila as a model system to study mitochondrial fatty acid metabolic disorders. Proceedings of the International Symposium on Mitochondrial Physiology and Pathology\u2014IUBMB Symposium S1\/2008, Bari, Italy."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"769","DOI":"10.1016\/j.cell.2008.06.037","article-title":"Chemical and biological approaches synergize to ameliorate protein-folding diseases","volume":"134","author":"Mu","year":"2008","journal-title":"Cell"},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"237","DOI":"10.1002\/bip.21319","article-title":"Identifying protein stabilizing ligands using GroEL","volume":"93","author":"Naik","year":"2010","journal-title":"Biopolymers"},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"2460","DOI":"10.2174\/1568026611212220002","article-title":"Protein misfolding in disease and small molecule therapies","volume":"12","author":"Gomes","year":"2012","journal-title":"Curr. Top. Med. Chem."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"943","DOI":"10.1016\/j.chembiol.2013.06.004","article-title":"Revertants, low temperature, and correctors reveal the mechanism of F508del-CFTR rescue by VX-809 and suggest multiple agents for full correction","volume":"20","author":"Farinha","year":"2013","journal-title":"Chem. Biol."},{"key":"ref_9","unstructured":"Henriques, B.J., Olsen, R.K.J., Bross, P., and Gomes, C.M. (2007, January 1\u20133). Role of cofactors in folding and kinetic stability in ETF, a protein involved in multiple acyl-coa dehydrogenase deficiency\u2014Oral communication. Proceedings of the Workshop\u2013Proteins in Health and Disease: From Structure to Function, Lisbon, Portugal."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"605","DOI":"10.1042\/BJ20060345","article-title":"Conformational stability of human frataxin and effect of Friedreich\u2019s ataxia-related mutations on protein folding","volume":"398","author":"Correia","year":"2006","journal-title":"Biochem. J."},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"3680","DOI":"10.1111\/j.1742-4658.2008.06512.x","article-title":"Dynamics, stability and iron-binding activity of frataxin clinical mutants","volume":"275","author":"Correia","year":"2008","journal-title":"FEBS J."},{"key":"ref_12","first-page":"270","article-title":"Natural history of muscle weakness in Friedreich\u2019s ataxia and its relation to loss of ambulation","volume":"311","author":"Beauchamp","year":"1995","journal-title":"Clin. Orthop. Relat. Res."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"200","DOI":"10.1002\/1531-8249(199902)45:2<200::AID-ANA10>3.0.CO;2-U","article-title":"Friedreich\u2019s ataxia: Point mutations and clinical presentation of compound heterozygotes","volume":"45","author":"Cossee","year":"1999","journal-title":"Ann. Neurol."},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"2253","DOI":"10.1016\/j.ccr.2012.04.004","article-title":"Metal ions as modulators of proteins conformation and misfolding in neurodegeneration","volume":"256","author":"Lear","year":"2012","journal-title":"Coord. Chem. Rev."},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"1007","DOI":"10.1016\/j.bbrc.2009.10.095","article-title":"The conserved trp155 in human frataxin as a hotspot for oxidative stress related chemical modifications","volume":"390","author":"Correia","year":"2009","journal-title":"Biochem. Biophys. Res. Commun."},{"key":"ref_16","unstructured":"Leal, S.S., and Gomes, C.M. (2008, January 10\u201313). On the relative contribution of ionic interactions over iron-sulfurclusters to ferredoxin stability. proceeding of the 1st Portuguese, Spanish-British joint Biophysics Congress, Lisbon, Portugal."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"2504","DOI":"10.2174\/1568026611212220006","article-title":"On the design of broad based screening assays to identify potential pharmacological chaperones of protein misfolding diseases","volume":"12","author":"Naik","year":"2012","journal-title":"Curr. Top. Med. Chem."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"25073","DOI":"10.1074\/jbc.273.39.25073","article-title":"Changing the nature of the initial chaperonin capture complex influences the substrate folding efficiency","volume":"273","author":"Voziyan","year":"1998","journal-title":"J. Biol. Chem."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"28677","DOI":"10.1074\/jbc.273.44.28677","article-title":"Partitioning of rhodanese onto GroEL. Chaperonin binds a reversibly oxidized form derived from the native protein","volume":"273","author":"Smith","year":"1998","journal-title":"J. Biol. Chem."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"9716","DOI":"10.1021\/bi00104a021","article-title":"Complex interactions between the chaperonin 60 molecular chaperone and dihydrofolate reductase","volume":"30","author":"Viitanen","year":"1991","journal-title":"Biochemistry"},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"9425","DOI":"10.1073\/pnas.93.18.9425","article-title":"A thermodynamic coupling mechanism for GroEL-mediated unfolding","volume":"93","author":"Walter","year":"1996","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"995","DOI":"10.1126\/science.1359644","article-title":"Prevention of protein denaturation under heat stress by the chaperonin hsp60","volume":"258","author":"Martin","year":"1992","journal-title":"Science"},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"21517","DOI":"10.1074\/jbc.270.37.21517","article-title":"Interactions between the GroE chaperonins and rhodanese. Multiple intermediates and release and rebinding","volume":"270","author":"Smith","year":"1995","journal-title":"J. Biol. Chem."},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"203","DOI":"10.1006\/jsbi.2001.4354","article-title":"Classification and reconstruction of a heterogeneous set of electron microscopic images: A case study of GroEL-substrate complexes","volume":"133","author":"Falke","year":"2001","journal-title":"J. Struct. Biol."},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"219","DOI":"10.1016\/j.jmb.2005.02.027","article-title":"The 13 angstroms structure of a chaperonin GroEL-protein substrate complex by cryo-electron microscopy","volume":"348","author":"Falke","year":"2005","journal-title":"J. Mol. Biol."},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"1461","DOI":"10.1002\/pro.2515","article-title":"Probing structurally altered and aggregated states of therapeutically relevant proteins using GroEL coupled to bio-layer interferometry","volume":"23","author":"Naik","year":"2014","journal-title":"Protein Sci."},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"1777","DOI":"10.1006\/jmbi.1998.2403","article-title":"The chaperonin groel binds to late-folding non-native conformations present in native Escherichia coli and murine dihydrofolate reductases","volume":"285","author":"Clark","year":"1999","journal-title":"J. Mol. Biol."},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"1586","DOI":"10.1021\/bi00220a020","article-title":"GroE facilitates refolding of citrate synthase by suppressing aggregation","volume":"30","author":"Buchner","year":"1991","journal-title":"Biochemistry"},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"884","DOI":"10.1038\/342884a0","article-title":"Reconstitution of active dimeric ribulose bisphosphate carboxylase from an unfolded state depends on two chaperonin proteins and Mg-ATP","volume":"342","author":"Goloubinoff","year":"1989","journal-title":"Nature"},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"512","DOI":"10.1006\/jmbi.1997.0969","article-title":"GroEL-mediated folding of structurally homologous dihydrofolate reductases","volume":"268","author":"Clark","year":"1997","journal-title":"J. Mol. Biol."},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"1553","DOI":"10.1002\/pro.5560040813","article-title":"Kinetics of interaction of partially folded proteins with a hydrophobic dye: Evidence that molten globule character is maximal in early folding intermediates","volume":"4","author":"Engelhard","year":"1995","journal-title":"Protein Sci."},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"181","DOI":"10.1016\/j.ab.2012.11.022","article-title":"In vivo detection and quantification of chemicals that enhance protein stability","volume":"434","author":"Hailu","year":"2013","journal-title":"Anal. Biochem."},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"157","DOI":"10.1002\/bip.20665","article-title":"From the test tube to the cell: Exploring the folding and aggregation of a beta-clam protein","volume":"88","author":"Ignatova","year":"2007","journal-title":"Biopolymers"},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"15866","DOI":"10.1074\/jbc.M110.107722","article-title":"Rescue of cystathionine beta-synthase (CBS) mutants with chemical chaperones: Purification and characterization of eight CBS mutant enzymes","volume":"285","author":"Majtan","year":"2010","journal-title":"J. Biol. Chem."},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"955","DOI":"10.1006\/jmbi.2001.4819","article-title":"The osmophobic effect: Natural selection of a thermodynamic force in protein folding","volume":"310","author":"Bolen","year":"2001","journal-title":"J. Mol. Biol."},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"141","DOI":"10.1016\/S0076-6879(98)90013-1","article-title":"Construction of single-ring and two-ring hybrid versions of bacterial chaperonin GroEL","volume":"290","author":"Horwich","year":"1998","journal-title":"Methods Enzymol."},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"312","DOI":"10.1016\/j.ymeth.2004.03.022","article-title":"Effects of naturally occurring osmolytes on protein stability and solubility: Issues important in protein crystallization","volume":"34","author":"Bolen","year":"2004","journal-title":"Methods"},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"744","DOI":"10.2174\/138920310794557727","article-title":"Compacting proteins: Pros and cons of osmolyte-induced folding","volume":"11","author":"Melo","year":"2010","journal-title":"Curr. Protein Pept. Sci."},{"key":"ref_39","doi-asserted-by":"crossref","first-page":"44541","DOI":"10.1074\/jbc.M106693200","article-title":"A comparison of the GroE chaperonin requirements for sequentially and structurally homologous malate dehydrogenases: The importance of folding kinetics and solution environment","volume":"276","author":"Tieman","year":"2001","journal-title":"J. Biol. Chem."},{"key":"ref_40","doi-asserted-by":"crossref","first-page":"2405","DOI":"10.1110\/ps.9.12.2405","article-title":"Chaperonin-assisted folding of glutamine synthetase under nonpermissive conditions: Off-pathway aggregation propensity does not determine the co-chaperonin requirement","volume":"9","author":"Voziyan","year":"2000","journal-title":"Protein Sci."},{"key":"ref_41","doi-asserted-by":"crossref","first-page":"17351","DOI":"10.1073\/pnas.0809794105","article-title":"Chaperonin chamber accelerates protein folding through passive action of preventing aggregation","volume":"105","author":"Apetri","year":"2008","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_42","doi-asserted-by":"crossref","first-page":"254","DOI":"10.1016\/0014-5793(91)80878-7","article-title":"Cooperativity in ATP hydrolysis by GroEL is increased by GroES","volume":"292","author":"Gray","year":"1991","journal-title":"FEBS Lett."},{"key":"ref_43","doi-asserted-by":"crossref","first-page":"1977","DOI":"10.1073\/pnas.93.5.1977","article-title":"Nucleotide binding-promoted conformational changes release a nonnative polypeptide from the Escherichia coli chaperonin GroEL","volume":"93","author":"Lin","year":"1996","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_44","doi-asserted-by":"crossref","first-page":"743","DOI":"10.1002\/pro.5560060401","article-title":"GroEL-mediated protein folding","volume":"6","author":"Fenton","year":"1997","journal-title":"Protein Sci."},{"key":"ref_45","doi-asserted-by":"crossref","first-page":"197","DOI":"10.1042\/BJ20091612","article-title":"Iron-binding activity in yeast frataxin entails a trade off with stability in the alpha1\/beta1 acidic ridge region","volume":"426","author":"Correia","year":"2010","journal-title":"Biochem. J."},{"key":"ref_46","doi-asserted-by":"crossref","first-page":"20071","DOI":"10.1016\/S0021-9258(20)80695-4","article-title":"Temperature-sensitive mutations and second-site suppressor substitutions affect folding of the p22 tailspike protein in vitro","volume":"268","author":"Mitraki","year":"1993","journal-title":"J. Biol. Chem."},{"key":"ref_47","doi-asserted-by":"crossref","first-page":"269","DOI":"10.1080\/10409230600846058","article-title":"The structure and function of frataxin","volume":"41","author":"Bencze","year":"2006","journal-title":"Crit. Rev. Biochem. Mol. Biol."},{"key":"ref_48","doi-asserted-by":"crossref","first-page":"7265","DOI":"10.1021\/bi200895k","article-title":"Structure-function analysis of Friedreich\u2019s ataxia mutants reveals determinants of frataxin binding and activation of the Fe-S assembly complex","volume":"50","author":"Winn","year":"2011","journal-title":"Biochemistry"},{"key":"ref_49","doi-asserted-by":"crossref","first-page":"4116","DOI":"10.1074\/jbc.M112.435263","article-title":"Missense mutations linked to friedreich ataxia have different but synergistic effects on mitochondrial frataxin isoforms","volume":"288","author":"Li","year":"2013","journal-title":"J. Biol. Chem."},{"key":"ref_50","doi-asserted-by":"crossref","first-page":"6511","DOI":"10.1021\/bi036049+","article-title":"The factors governing the thermal stability of frataxin orthologues: How to increase a protein\u2019s stability","volume":"43","author":"Adinolfi","year":"2004","journal-title":"Biochemistry"},{"key":"ref_51","doi-asserted-by":"crossref","first-page":"695","DOI":"10.1016\/S0969-2126(00)00158-1","article-title":"Towards a structural understanding of friedreich\u2019s ataxia: The solution structure of frataxin","volume":"8","author":"Musco","year":"2000","journal-title":"Structure"},{"key":"ref_52","doi-asserted-by":"crossref","first-page":"5893","DOI":"10.1021\/bi953051v","article-title":"Determination of regions in the dihydrofolate reductase structure that interact with the molecular chaperonin GroEL","volume":"35","author":"Clark","year":"1996","journal-title":"Biochemistry"},{"key":"ref_53","doi-asserted-by":"crossref","first-page":"581","DOI":"10.1146\/annurev.biochem.67.1.581","article-title":"Structure and function in GroEL-mediated protein folding","volume":"67","author":"Sigler","year":"1998","journal-title":"Annu. Rev. Biochem."},{"key":"ref_54","doi-asserted-by":"crossref","first-page":"3794","DOI":"10.1021\/ja8049063","article-title":"High-throughput thermal scanning: A general, rapid dye-binding thermal shift screen for protein engineering","volume":"131","author":"Lavinder","year":"2009","journal-title":"J. Am. Chem. Soc."},{"key":"ref_55","doi-asserted-by":"crossref","first-page":"217","DOI":"10.1039\/B814377C","article-title":"High throughput methods of assessing protein stability and aggregation","volume":"5","author":"Senisterra","year":"2009","journal-title":"Mol. Biosyst."},{"key":"ref_56","doi-asserted-by":"crossref","first-page":"271","DOI":"10.1006\/jmbi.1998.1760","article-title":"Conformational stability and thermodynamics of folding of ribonucleases SA, SA2 and SA3","volume":"279","author":"Pace","year":"1998","journal-title":"J. Mol. Biol."},{"key":"ref_57","doi-asserted-by":"crossref","first-page":"959","DOI":"10.1146\/annurev.biochem.052308.114844","article-title":"Biological and chemical approaches to diseases of proteostasis deficiency","volume":"78","author":"Powers","year":"2009","journal-title":"Annu. Rev. Biochem."}],"container-title":["Biomolecules"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/2218-273X\/4\/4\/956\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,11]],"date-time":"2025-10-11T21:17:10Z","timestamp":1760217430000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/2218-273X\/4\/4\/956"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2014,10,20]]},"references-count":57,"journal-issue":{"issue":"4","published-online":{"date-parts":[[2014,12]]}},"alternative-id":["biom4040956"],"URL":"https:\/\/doi.org\/10.3390\/biom4040956","relation":{},"ISSN":["2218-273X"],"issn-type":[{"type":"electronic","value":"2218-273X"}],"subject":[],"published":{"date-parts":[[2014,10,20]]}}}