{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,1,9]],"date-time":"2026-01-09T12:42:21Z","timestamp":1767962541394,"version":"3.49.0"},"reference-count":56,"publisher":"MDPI AG","issue":"9","license":[{"start":{"date-parts":[[2013,8,22]],"date-time":"2013-08-22T00:00:00Z","timestamp":1377129600000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/3.0\/"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["IJMS"],"abstract":"<jats:p>We use molecular dynamics simulations of a full atomistic G\u014d model to explore the impact of selected DE-loop mutations (D59P and W60C) on the folding space of protein human \u03b22-microglobulin (H\u03b22m), the causing agent of dialysis-related amyloidosis, a conformational disorder characterized by the deposition of insoluble amyloid fibrils in the osteoarticular system. Our simulations replicate the effect of mutations on the thermal stability that is observed in experiments in vitro. Furthermore, they predict the population of a partially folded state, with 60% of native internal free energy, which is akin to a molten globule. In the intermediate state, the solvent accessible surface area increases up to  40 times relative to the native state in 38% of the hydrophobic core residues, indicating that the identified species has aggregation potential. The intermediate state preserves the disulfide bond established between residue Cys25 and residue Cys80, which helps maintain the integrity of the core region, and is characterized by having two unstructured termini. The movements of the termini dominate the essential modes of the intermediate state, and exhibit the largest displacements in the D59P mutant, which is the most aggregation prone variant. PROPKA predictions of pKa suggest that the population of the intermediate state may be enhanced at acidic pH explaining the larger amyloidogenic potential observed  in vitro at low pH for the WT protein and mutant forms.<\/jats:p>","DOI":"10.3390\/ijms140917256","type":"journal-article","created":{"date-parts":[[2013,8,22]],"date-time":"2013-08-22T11:56:40Z","timestamp":1377172600000},"page":"17256-17278","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":18,"title":["Assessing the Effect of Loop Mutations in the Folding Space of \u03b22-Microglobulin with Molecular Dynamics Simulations"],"prefix":"10.3390","volume":"14","author":[{"given":"S\u00edlvia","family":"Est\u00e1cio","sequence":"first","affiliation":[{"name":"Centre for Condensed Matter Physics, University of Lisbon, Av. Prof. Gama Pinto 2,  Lisboa 1649-003, Portugal"},{"name":"Department of Physics, University of Lisbon, Av. Prof. Gama Pinto 2, Lisboa 1649-003, Portugal"}]},{"given":"Eugene","family":"Shakhnovich","sequence":"additional","affiliation":[{"name":"Department of Chemistry and Chemical Biology, Harvard University, 12 Oxford Street, Cambridge, MA 02138, USA"}]},{"given":"Patr\u00edcia","family":"Fa\u00edsca","sequence":"additional","affiliation":[{"name":"Centre for Condensed Matter Physics, University of Lisbon, Av. Prof. Gama Pinto 2,  Lisboa 1649-003, Portugal"},{"name":"Department of Physics, University of Lisbon, Av. Prof. Gama Pinto 2, Lisboa 1649-003, Portugal"}]}],"member":"1968","published-online":{"date-parts":[[2013,8,22]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"9771","DOI":"10.1073\/pnas.152337399","article-title":"Crystal structure of monomeric human \u03b2-2-microglobulin reveals clues to its amyloidogenic properties","volume":"99","author":"Trinh","year":"2002","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"101","DOI":"10.1016\/S0006-291X(03)00543-6","article-title":"Amyloidogenic synthetic peptides of \u03b22-microglobulin\u2014A role of the disulfide bond","volume":"304","author":"Hasegawa","year":"2003","journal-title":"Biochem. Biophys. Res. Commun"},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"258","DOI":"10.1016\/j.jmb.2007.12.002","article-title":"Thiol compounds inhibit the formation of amyloid fibrils by \u03b22-microglobulin at neutral pH","volume":"376","author":"Yamamoto","year":"2008","journal-title":"J. Mol. Biol"},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"117","DOI":"10.1046\/j.1525-139x.2001.00030.x","article-title":"Dynamic of \u03b22-microglobulin fibril formation and reabsorption: The role of proteolysis","volume":"14","author":"Bellotti","year":"2001","journal-title":"Semin. Dial"},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"92","DOI":"10.1016\/j.bbapap.2005.09.002","article-title":"From chance to frequent encounters: Origins of \u03b22-microglobulin fibrillogenesis","volume":"1753","author":"Eakin","year":"2005","journal-title":"BBA-Proteins Proteom"},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"2623","DOI":"10.1016\/j.febslet.2009.05.005","article-title":"Glimpses of the molecular mechanisms of \u03b22-microglobulin fibril formation in vitro: Aggregation on a complex energy landscape","volume":"583","author":"Platt","year":"2009","journal-title":"FEBS Lett"},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"943","DOI":"10.1016\/S0022-2836(03)00687-9","article-title":"A systematic investigation into the effect of protein destabilisation on beta 2-microglobulin amyloid formation","volume":"330","author":"Smith","year":"2003","journal-title":"J. Mol. Biol"},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"32843","DOI":"10.1074\/jbc.M506501200","article-title":"Ultrasonication-induced amyloid fibril formation of \u03b22-microglobulin","volume":"280","author":"Ohhashi","year":"2005","journal-title":"J. Biol. Chem"},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"965","DOI":"10.1038\/nsmb.1483","article-title":"A regulatable switch mediates self-association in an immunoglobulin fold","volume":"15","author":"Calabrese","year":"2008","journal-title":"Nat. Struct. Mol. Biol"},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"1783","DOI":"10.1002\/rcm.6282","article-title":"The role of conformational flexibility in \u03b22-microglobulin amyloid fibril formation at neutral pH","volume":"26","author":"Hodkinson","year":"2012","journal-title":"Rapid Commun. Mass Spectrom"},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"379","DOI":"10.1006\/jmbi.2000.4478","article-title":"Detection of two partially structured species in the folding process of the amyloidogenic protein [beta]2-microglobulin","volume":"307","author":"Chiti","year":"2001","journal-title":"J. Mol. Biol."},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"46714","DOI":"10.1074\/jbc.M107040200","article-title":"A partially structured species of [beta]2-microglobulin is significantly populated under physiological conditions and involved in fibrillogenesis","volume":"276","author":"Chiti","year":"2001","journal-title":"J. Biol. Chem"},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"195","DOI":"10.1038\/nsmb1058","article-title":"Amyloid formation under physiological conditions proceeds via a native-like folding intermediate","volume":"13","author":"Jahn","year":"2006","journal-title":"Nat. Struct. Mol. Biol"},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"1312","DOI":"10.1016\/j.jmb.2009.01.013","article-title":"A generic mechanism of \u03b22-microglobulin amyloid assembly at neutral pH involving a specific proline switch","volume":"386","author":"Eichner","year":"2009","journal-title":"J. Mol. Biol"},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"831","DOI":"10.1110\/ps.9.5.831","article-title":"Removal of the N-terminal hexapeptide from human \u03b22-microglobulin facilitates protein aggregation and fibril formation","volume":"9","author":"Esposito","year":"2000","journal-title":"Protein Sci"},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"161","DOI":"10.1016\/j.molcel.2010.11.028","article-title":"Conformational conversion during amyloid formation at atomic resolution","volume":"41","author":"Eichner","year":"2011","journal-title":"Mol. Cell"},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"776","DOI":"10.1016\/j.jmb.2009.04.042","article-title":"Competition between intramolecular and intermolecular interactions in an amyloid-forming protein","volume":"389","author":"Routledge","year":"2009","journal-title":"J. Mol. Biol"},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"1522","DOI":"10.1021\/bi901748h","article-title":"Structure of the preamyloid dimer of \u03b2-2-microglobulin from covalent labeling and mass spectrometry","volume":"49","author":"Mendoza","year":"2010","journal-title":"Biochemistry"},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"1131","DOI":"10.1111\/j.1742-4658.2012.08510.x","article-title":"A recurrent D-strand association interface is observed in \u03b2-2 microglobulin oligomers","volume":"279","author":"Colombo","year":"2012","journal-title":"FEBS J"},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"887","DOI":"10.1016\/j.jmb.2008.03.002","article-title":"The controlling roles of Trp60 and Trp95 in \u03b22-microglobulin function, folding and amyloid aggregation properties","volume":"378","author":"Esposito","year":"2008","journal-title":"J. Mol. Biol"},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"1386","DOI":"10.1002\/pro.419","article-title":"DE-loop mutations affect beta2 microglobulin stability, oligomerization, and the low-pH unfolded form","volume":"19","author":"Santambrogio","year":"2010","journal-title":"Protein Sci"},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"146","DOI":"10.1016\/j.bbrc.2008.09.108","article-title":"DE loop mutations affect \u03b22-microglobulin stability and amyloid aggregation","volume":"377","author":"Ricagno","year":"2008","journal-title":"Biochem. Biophys. Res. Commun"},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"159","DOI":"10.1186\/1471-2148-11-159","article-title":"The two tryptophans of beta2-microglobulin have distinct roles in function and folding and might represent two independent responses to evolutionary pressure","volume":"11","author":"Raimondi","year":"2011","journal-title":"BMC Evol. Biol"},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"31061","DOI":"10.1074\/jbc.M605358200","article-title":"Conformation of amyloid fibrils of \u03b22-microglobulin probed by tryptophan mutagenesis","volume":"281","author":"Kihara","year":"2006","journal-title":"J. Biol. Chem"},{"key":"ref_25","doi-asserted-by":"crossref","unstructured":"Est\u00e1cio, S.G., Fernandes, C.S., Krobath, H., Fa\u00edsca, P.F.N., and Shakhnovich, E.I. (2012). Robustness of atomistic G\u014d models in predicting native-like folding intermediates. J. Chem. Phys., 137.","DOI":"10.1063\/1.4747492"},{"key":"ref_26","unstructured":"Esposito, G., and Bellotti, V. (2010). Protein Misfolding Diseases, John Wiley & Sons, Inc."},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"2754","DOI":"10.1073\/pnas.052706099","article-title":"Natural \u03b2-sheet proteins use negative design to avoid edge-to-edge aggregation","volume":"99","author":"Richardson","year":"2002","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"721","DOI":"10.1038\/1418","article-title":"Obligatory steps in protein folding and the conformational diversity of the transition state","volume":"5","author":"Martinez","year":"1998","journal-title":"Nat. Struct. Mol. Biol"},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"21","DOI":"10.1016\/0014-5793(83)80010-6","article-title":"\u201cMolten-globule state\u201d: A compact form of globular proteins with mobile side-chains","volume":"164","author":"Ohgushi","year":"1983","journal-title":"FEBS Lett"},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"43","DOI":"10.1007\/0-387-25919-8_3","article-title":"Relative Importance of Hydrophobicity, Net Charge, and Secondary Structure Propensities in Protein Aggregation","volume":"4","author":"Chiti","year":"2006","journal-title":"Protein Misfolding, Aggregation, and Conformational Diseases\u2014Protein Reviews"},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"326","DOI":"10.1038\/nsb791","article-title":"Structural properties of an amyloid precursor of [beta]2-microglobulin","volume":"9","author":"McParland","year":"2002","journal-title":"Nat. Struct. Biol"},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"273","DOI":"10.1016\/j.jmb.2012.11.002","article-title":"The molten globule of \u03b22-microglobulin accumulated at pH 4 and its role in protein folding","volume":"425","author":"Mukaiyama","year":"2013","journal-title":"J. Mol. Biol"},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"257","DOI":"10.1016\/j.jmb.2012.11.004","article-title":"Native-state heterogeneity of \u03b22-microglobulin as revealed by kinetic folding and real-time NMR experiments","volume":"425","author":"Mukaiyama","year":"2013","journal-title":"J. Mol. Biol"},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"8735","DOI":"10.1021\/bi000276j","article-title":"Partially unfolded states of \u03b22-microglobulin and amyloid formation in vitro","volume":"39","author":"McParland","year":"2000","journal-title":"Biochemistry"},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"279","DOI":"10.1016\/j.jmb.2004.11.035","article-title":"Dynamics in the unfolded state of [beta]2-microglobulin studied by NMR","volume":"346","author":"Platt","year":"2005","journal-title":"J. Mol. Biol"},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"704","DOI":"10.1002\/prot.20660","article-title":"Very fast empirical prediction and rationalization of protein pKa values","volume":"61","author":"Li","year":"2005","journal-title":"Proteins: Struct. Funct. Bioinf"},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"705","DOI":"10.1016\/j.jmb.2012.06.020","article-title":"Identification of a conserved aggregation-prone intermediate state in the folding pathways of Spc-SH3 amyloidogenic variants","volume":"422","author":"Krobath","year":"2012","journal-title":"J. Mol. Biol"},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"677","DOI":"10.1002\/pro.2053","article-title":"Is there an en route folding intermediate for cold shock proteins?","volume":"21","author":"Huang","year":"2012","journal-title":"Protein Sci"},{"key":"ref_39","doi-asserted-by":"crossref","first-page":"215","DOI":"10.1006\/jmbi.1999.2949","article-title":"Hierarchy of structure loss in MD simulations of src SH3 domain unfolding","volume":"291","author":"Tsai","year":"1999","journal-title":"J. Mol. Biol"},{"key":"ref_40","doi-asserted-by":"crossref","first-page":"991","DOI":"10.1002\/bip.1981.360200512","article-title":"Noninteracting local-structure model of folding and unfolding transition in globular proteins. I. Formulation","volume":"20","author":"Abe","year":"1981","journal-title":"Biopolymers"},{"key":"ref_41","doi-asserted-by":"crossref","first-page":"79","DOI":"10.1006\/jmbi.2001.4586","article-title":"The folding thermodynamics and kinetics of crambin using an all-atom Monte Carlo simulation","volume":"308","author":"Shimada","year":"2001","journal-title":"J. Mol. Biol"},{"key":"ref_42","doi-asserted-by":"crossref","first-page":"577","DOI":"10.1016\/S1359-0278(98)00072-8","article-title":"Discrete molecular dynamics studies of the folding of a protein-like model","volume":"3","author":"Dokholyan","year":"1998","journal-title":"Fold. Des"},{"key":"ref_43","doi-asserted-by":"crossref","first-page":"16","DOI":"10.1006\/jcph.1996.5510","article-title":"Molecular dynamics for polymeric fluids using discontinuous potentials","volume":"134","author":"Smith","year":"1997","journal-title":"J. Comput. Phys"},{"key":"ref_44","doi-asserted-by":"crossref","first-page":"141","DOI":"10.1016\/S0009-2614(99)01123-9","article-title":"Replica-exchange molecular dynamics method for protein folding","volume":"314","author":"Sugita","year":"1999","journal-title":"Chem. Phys. Lett"},{"key":"ref_45","doi-asserted-by":"crossref","first-page":"26","DOI":"10.1021\/ct0502864","article-title":"Use of the weighted histogram analysis method for the analysis of simulated and parallel tempering simulations","volume":"3","author":"Chodera","year":"2007","journal-title":"J. Chem. Theor. Comput"},{"key":"ref_46","doi-asserted-by":"crossref","first-page":"377","DOI":"10.1016\/j.jmgm.2003.12.005","article-title":"MMTSB Tool Set: enhanced sampling and multiscale modeling methods for applications in structural biology","volume":"22","author":"Feig","year":"2004","journal-title":"J. Mol. Graph. Model."},{"key":"ref_47","doi-asserted-by":"crossref","first-page":"845","DOI":"10.1093\/bioinformatics\/btt055","article-title":"GROMACS 4.5: A high-throughput and highly parallel open source molecular simulation toolkit","volume":"29","author":"Pronk","year":"2013","journal-title":"Bioinformatics"},{"key":"ref_48","doi-asserted-by":"crossref","first-page":"362","DOI":"10.1126\/science.1214203","article-title":"Structure of an intermediate state in protein folding and aggregation","volume":"336","author":"Neudecker","year":"2012","journal-title":"Science"},{"key":"ref_49","doi-asserted-by":"crossref","first-page":"15","DOI":"10.1038\/nchembio.131","article-title":"Amyloid formation by globular proteins under native conditions","volume":"5","author":"Chiti","year":"2009","journal-title":"Nat. Chem. Biol"},{"key":"ref_50","doi-asserted-by":"crossref","first-page":"935","DOI":"10.1016\/S0022-2836(03)00688-0","article-title":"Role of the N and C-terminal strands of [beta]2-microglobulin in amyloid formation at neutral pH","volume":"330","author":"Jones","year":"2003","journal-title":"J. Mol. Biol"},{"key":"ref_51","doi-asserted-by":"crossref","first-page":"1235","DOI":"10.1002\/prot.21655","article-title":"Solvent effects in the slow dynamics of proteins","volume":"70","author":"Hinsen","year":"2008","journal-title":"Proteins Struct. Funct. Bioinf"},{"key":"ref_52","doi-asserted-by":"crossref","first-page":"1544","DOI":"10.1126\/science.2218495","article-title":"Structural characterization of a partly folded apomyoglobin intermediate","volume":"249","author":"Hughson","year":"1990","journal-title":"Science"},{"key":"ref_53","first-page":"676","article-title":"Hydrophobic core variant ubiquitin forms a molten globule conformation at acidic pH","volume":"37","author":"Park","year":"2004","journal-title":"J. Biochem. Mol. Biol"},{"key":"ref_54","doi-asserted-by":"crossref","first-page":"W522","DOI":"10.1093\/nar\/gkm276","article-title":"PDB2PQR: Expanding and upgrading automated preparation of biomolecular structures for molecular simulations","volume":"35","author":"Dolinsky","year":"2004","journal-title":"Nucleic Acids Res"},{"key":"ref_55","doi-asserted-by":"crossref","first-page":"10037","DOI":"10.1073\/pnas.181342398","article-title":"Electrostatics of nanosystems: Application to microtubules and the ribosome","volume":"98","author":"Baker","year":"2001","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_56","doi-asserted-by":"crossref","first-page":"33","DOI":"10.1016\/0263-7855(96)00018-5","article-title":"VMD: Visual molecular dynamics","volume":"14","author":"Humphrey","year":"1996","journal-title":"J. Mol. Graph"}],"container-title":["International Journal of Molecular Sciences"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1422-0067\/14\/9\/17256\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,11]],"date-time":"2025-10-11T21:48:50Z","timestamp":1760219330000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1422-0067\/14\/9\/17256"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2013,8,22]]},"references-count":56,"journal-issue":{"issue":"9","published-online":{"date-parts":[[2013,9]]}},"alternative-id":["ijms140917256"],"URL":"https:\/\/doi.org\/10.3390\/ijms140917256","relation":{},"ISSN":["1422-0067"],"issn-type":[{"value":"1422-0067","type":"electronic"}],"subject":[],"published":{"date-parts":[[2013,8,22]]}}}