{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2025,7,30]],"date-time":"2025-07-30T11:14:59Z","timestamp":1753874099451,"version":"3.41.2"},"reference-count":55,"publisher":"AIP Publishing","issue":"4","license":[{"start":{"date-parts":[[2023,7,18]],"date-time":"2023-07-18T00:00:00Z","timestamp":1689638400000},"content-version":"vor","delay-in-days":17,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"},{"start":{"date-parts":[[2023,7,18]],"date-time":"2023-07-18T00:00:00Z","timestamp":1689638400000},"content-version":"tdm","delay-in-days":17,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/100000057","name":"National Institute of General Medical Sciences","doi-asserted-by":"publisher","award":["R01-40906"],"award-info":[{"award-number":["R01-40906"]}],"id":[{"id":"10.13039\/100000057","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/100000057","name":"National Institute of General Medical Sciences","doi-asserted-by":"publisher","award":["R01-GM114015"],"award-info":[{"award-number":["R01-GM114015"]}],"id":[{"id":"10.13039\/100000057","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/100000057","name":"National Institute of General Medical Sciences","doi-asserted-by":"publisher","award":["R01-GM064803"],"award-info":[{"award-number":["R01-GM064803"]}],"id":[{"id":"10.13039\/100000057","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/100000057","name":"National Institute of General Medical Sciences","doi-asserted-by":"publisher","award":["R01-GM123247"],"award-info":[{"award-number":["R01-GM123247"]}],"id":[{"id":"10.13039\/100000057","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/100000002","name":"Yes, funding has been received from NIH","doi-asserted-by":"publisher","id":[{"id":"10.13039\/100000002","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":["pubs.aip.org"],"crossmark-restriction":true},"short-container-title":[],"published-print":{"date-parts":[[2023,7,1]]},"abstract":"<jats:p>Mechanistic studies of Geobacillus stearothermophilus tryptophanyl-tRNA synthetase (TrpRS) afford an unusually detailed description\u2014the escapement mechanism\u2014for the distinct steps coupling catalysis to domain motion, efficiently converting the free energy of ATP hydrolysis into biologically useful alternative forms of information and work. Further elucidation of the escapement mechanism requires understanding thermodynamic linkages between domain configuration and conformational stability. To that end, we compare experimental thermal melting of fully liganded and apo TrpRS with a computational simulation of the melting of its fully liganded form. The simulation also provides important structural cameos at successively higher temperatures, enabling more confident interpretation. Experimental and simulated melting both proceed through a succession of three transitions at successively higher temperature. The low-temperature transition occurs at approximately the growth temperature of the organism and so may be functionally relevant but remains too subtle to characterize structurally. Structural metrics from the simulation imply that the two higher-temperature transitions entail forming a molten globular state followed by unfolding of secondary structures. Ligands that stabilize the enzyme in a pre-transition (PreTS) state compress the temperature range over which these transitions occur and sharpen the transitions to the molten globule and fully denatured states, while broadening the low-temperature transition. The experimental enthalpy changes provide a key parameter necessary to convert changes in melting temperature of combinatorial mutants into mutationally induced conformational free energy changes. The TrpRS urzyme, an excerpted model representing an early ancestral form, containing virtually the entire catalytic apparatus, remains largely intact at the highest simulated temperatures.<\/jats:p>","DOI":"10.1063\/4.0000181","type":"journal-article","created":{"date-parts":[[2023,7,18]],"date-time":"2023-07-18T17:35:55Z","timestamp":1689701755000},"update-policy":"https:\/\/doi.org\/10.1063\/aip-crossmark-policy-page","source":"Crossref","is-referenced-by-count":1,"title":["Microcalorimetry reveals multi-state thermal denaturation of <i>G. stearothermophilus<\/i> tryptophanyl-tRNA synthetase"],"prefix":"10.1063","volume":"10","author":[{"ORCID":"https:\/\/orcid.org\/0000-0001-8874-9988","authenticated-orcid":false,"given":"Srinivas Niranj","family":"Chandrasekaran","sequence":"first","affiliation":[{"name":"Imaging Platform, Broad Institute of MIT and Harvard, Cambridge 1 , Massachusetts 02142, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Jhuma","family":"Das","sequence":"additional","affiliation":[{"name":"Cystic Fibrosis and Pulmonary Diseases Research and Treatment Center, University of North Carolina at Chapel Hill, Chapel Hill 2 , North Carolina 27599, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-8225-4025","authenticated-orcid":false,"given":"Nikolay V.","family":"Dokholyan","sequence":"additional","affiliation":[{"name":"Department of Pharmacology and Biochemistry and Molecular Biology, Penn State College of Medicine 3 , Hershey, Pennsylvania 17033, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-2653-4452","authenticated-orcid":false,"suffix":"Jr.","given":"Charles W.","family":"Carter","sequence":"additional","affiliation":[{"name":"Department of Biophysics and Biochemistry, University of North Carolina at Chapel Hill 4 , Chapel Hill, North Carolina 27599, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"317","published-online":{"date-parts":[[2023,7,18]]},"reference":[{"key":"2023071817355018900_c1","doi-asserted-by":"publisher","first-page":"032101","DOI":"10.1063\/1.4974218","article-title":"Combining multi-mutant and modular thermodynamic cycles to measure energetic coupling networks in enzyme catalysis","volume":"4","year":"2017","journal-title":"Struct. Dyn."},{"key":"2023071817355018900_c2","doi-asserted-by":"publisher","first-page":"433","DOI":"10.1146\/annurev-biophys-070816-033811","article-title":"High-dimensional mutant and modular thermodynamic cycles, molecular switching, and free energy transduction","volume":"46","year":"2017","journal-title":"Annu. Rev. Biophys."},{"key":"2023071817355018900_c3","doi-asserted-by":"publisher","first-page":"128","DOI":"10.1016\/j.str.2011.10.020","article-title":"A master switch couples Mg2+-assisted catalysis to domain motion in B. stearothermophilus tryptophanyl-tRNA synthetase","volume":"20","year":"2012","journal-title":"Structure"},{"key":"2023071817355018900_c4","doi-asserted-by":"publisher","first-page":"710","DOI":"10.1002\/prot.25856","article-title":"Escapement mechanisms: Efficient free energy transduction by reciprocally-coupled gating","volume":"88","year":"2019","journal-title":"Proteins"},{"key":"2023071817355018900_c5","doi-asserted-by":"publisher","first-page":"952","DOI":"10.1016\/j.str.2009.05.007","article-title":"Mg2+-assisted catalysis by B. stearothermophilus TrpRS is promoted by allosteric effects","volume":"17","year":"2009","journal-title":"Structure"},{"key":"2023071817355018900_c6","doi-asserted-by":"publisher","first-page":"1488","DOI":"10.1021\/ja076557x","article-title":"Mg2+-free B. stearothermophilus tryptophanyl-tRNA synthetase activates tryptophan with a major fraction of the overall rate enhancement","volume":"130","year":"2008","journal-title":"J. Am. Chem. Soc."},{"key":"2023071817355018900_c7","doi-asserted-by":"publisher","first-page":"012101","DOI":"10.1063\/1.4941599","article-title":"A modified PATH algorithm rapidly generates transition states comparable to those found by other well established algorithms","volume":"3","year":"2016","journal-title":"Struct. Dyn."},{"key":"2023071817355018900_c8","doi-asserted-by":"publisher","first-page":"1","DOI":"10.1016\/j.physrep.2017.02.001","article-title":"Escapement mechanisms and the conversion of disequilibria; the engines of creation","volume":"677","year":"2017","journal-title":"Phys. Rep."},{"key":"2023071817355018900_c9","doi-asserted-by":"publisher","first-page":"1700179","DOI":"10.1002\/bies.201700179","article-title":"Frankenstein or a submarine alkaline vent: Who is responsible for abiogenesis? Part 1: What is life\u2013that it might create itself?","volume":"40","year":"2018","journal-title":"BioEssays"},{"key":"2023071817355018900_c10","doi-asserted-by":"publisher","first-page":"265","DOI":"10.3390\/biom11020265","article-title":"Reciprocally-coupled gating: Strange loops in bioenergetics, genetics, and catalysis","volume":"11","year":"2021","journal-title":"Biomolecules"},{"key":"2023071817355018900_c11","doi-asserted-by":"publisher","first-page":"2459","DOI":"10.1002\/pro.5560031232","article-title":"Reaction mechanisms, catalysis, and movement","volume":"3","year":"1994","journal-title":"Prot. Sci."},{"key":"2023071817355018900_c12","doi-asserted-by":"publisher","first-page":"145","DOI":"10.1016\/S0076-6879(89)71010-7","article-title":"Utilization of binding energy and coupling rules for active transport and other coupled vectorial processes","volume":"171","year":"1989","journal-title":"Methods Enzymol."},{"key":"2023071817355018900_c13","doi-asserted-by":"publisher","first-page":"13861","DOI":"10.1073\/pnas.0504026102","article-title":"Initiation of the power stroke in muscle: Insights from the phosphate analog AlF4","volume":"102","year":"2005","journal-title":"Proc. Natl. Acad. Sci. U. S. A."},{"key":"2023071817355018900_c14","doi-asserted-by":"publisher","first-page":"989","DOI":"10.1016\/j.tibs.2016.09.006","article-title":"How myosin generates force on actin filaments","volume":"41","year":"2016","journal-title":"Trends Biochem. Sci."},{"key":"2023071817355018900_c15","doi-asserted-by":"publisher","first-page":"1159","DOI":"10.1016\/j.jmb.2006.06.078","article-title":"Computational studies of tryptophanyl-tRNA synthetase: Activation of ATP by induced-fit","volume":"362","year":"2006","journal-title":"J. Mol. Biol."},{"key":"2023071817355018900_c16","doi-asserted-by":"publisher","first-page":"709","DOI":"10.1093\/bioinformatics\/btq039","article-title":"Six rossmannoid folds, including the class I aminoacyl-tRNA synthetases, share a partial core with the anticodon-binding domain of a class II aminoacyl-tRNA synthetase","volume":"26","year":"2010","journal-title":"Bioinformatics"},{"key":"2023071817355018900_c17","doi-asserted-by":"publisher","first-page":"1272","DOI":"10.1016\/j.str.2007.08.010","article-title":"A conformational transition state accompanies tryptophan activation by B. stearothermphilus tryptophanyl-tRNA synthetase","volume":"15","year":"2007","journal-title":"Structure"},{"key":"2023071817355018900_c18","doi-asserted-by":"publisher","first-page":"509","DOI":"10.1016\/S0076-6879(03)74022-1","article-title":"Simplicial neighborhood analysis of protein packing (SNAPP): A computational geometry approach to studying proteins","volume":"374","year":"2003","journal-title":"Methods Enzymol."},{"key":"2023071817355018900_c19","doi-asserted-by":"publisher","first-page":"625","DOI":"10.1006\/jmbi.2001.4906","article-title":"Four-body potentials reveal protein-specific correlations to stability changes caused by hydrophobic core mutations","volume":"311","year":"2001","journal-title":"J. Mol. Biol."},{"key":"2023071817355018900_c20","doi-asserted-by":"publisher","first-page":"032103","DOI":"10.1063\/1.4976142","article-title":"Adding torsional interaction terms to the anisotropic network model improves the PATH performance, enabling detailed comparison with experimental rate data","volume":"4","year":"2017","journal-title":"Struct. Dyn."},{"key":"2023071817355018900_c21","doi-asserted-by":"publisher","first-page":"W235","DOI":"10.1093\/nar\/gkl163","article-title":"RosettaDesign server for protein design","volume":"34","year":"2006","journal-title":"Nucleic Acids Res."},{"key":"2023071817355018900_c22","doi-asserted-by":"publisher","first-page":"34736","DOI":"10.1074\/jbc.M113.510958","article-title":"Full implementation of the genetic code by tryptophanyl-tRNA synthetase requires intermodular coupling","volume":"288","year":"2013","journal-title":"J. Biol. Chem."},{"year":"2023","key":"2023071817355018900_c23","article-title":"The LeuAC urzyme catalyzes aminoacylation of tRNALeu minihelix using leucine and ATP"},{"key":"2023071817355018900_c24","doi-asserted-by":"publisher","first-page":"39","DOI":"10.1016\/S0022-2836(02)01156-7","article-title":"Interconversion of ATP binding and conformational free energies by tryptophanyl-tRNA synthetase: Structures of ATP bound to open and closed, pre-transition conformations","volume":"325","year":"2003","journal-title":"J. Mol. Biol."},{"key":"2023071817355018900_c25","doi-asserted-by":"publisher","first-page":"218","DOI":"10.1110\/ps.9.2.218","article-title":"2.9 \u00c5 crystal structure of ligand-free tryptophanyl-tRNA synthetase: Domain movements fragment the adenine nucleotide binding site","volume":"9","year":"2000","journal-title":"Protein Sci."},{"year":"2023","key":"2023071817355018900_c26","article-title":"High-throughput thermal denaturation of tryptophanyl-tRNA synthetase combinatorial mutants reveals high-order energetic coupling determinants of conformational stability"},{"key":"2023071817355018900_c27","doi-asserted-by":"publisher","first-page":"e1004359","DOI":"10.1371\/journal.pcbi.1004359","article-title":"ApoE4-specific misfolded intermediate identified by molecular dynamics simulations","volume":"11","year":"2015","journal-title":"PLoS Comput. Biol."},{"key":"2023071817355018900_c28","doi-asserted-by":"publisher","first-page":"1183","DOI":"10.1006\/jmbi.1999.3534","article-title":"Identifying the protein folding nucleus using molecular dynamics","volume":"296","year":"2000","journal-title":"J. Mol. Biol."},{"key":"2023071817355018900_c29","doi-asserted-by":"publisher","first-page":"8375","DOI":"10.1021\/jp2114576","article-title":"Discrete molecular dynamics: An efficient and versatile simulation method for fine protein characterization","volume":"116","year":"2012","journal-title":"J. Phys. Chem. B"},{"key":"2023071817355018900_c30","doi-asserted-by":"publisher","first-page":"1010","DOI":"10.1016\/j.str.2008.03.013","article-title":"Ab-initio folding of proteins with all-atom discrete molecular dynamics","volume":"16","year":"2008","journal-title":"Structure"},{"key":"2023071817355018900_c31","doi-asserted-by":"publisher","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","year":"1998","journal-title":"Folding Des."},{"key":"2023071817355018900_c32","doi-asserted-by":"publisher","first-page":"8637","DOI":"10.1073\/pnas.122076099","article-title":"Topological determinants of protein folding","volume":"99","year":"2002","journal-title":"Proc. Natl. Acad. Sci. U. S. A."},{"key":"2023071817355018900_c33","doi-asserted-by":"publisher","first-page":"2384","DOI":"10.1063\/1.439486","article-title":"Molecular dynamics simulations at constant pressure and\/or temperature","volume":"72","year":"1980","journal-title":"J. Chem. Phys."},{"key":"2023071817355018900_c34","doi-asserted-by":"publisher","first-page":"139","DOI":"10.1016\/S0959-440X(00)00063-4","article-title":"Effective energy functions for protein structure prediction","volume":"10","year":"2000","journal-title":"Curr. Opin. Struct. Biol."},{"key":"2023071817355018900_c35","doi-asserted-by":"publisher","first-page":"3296","DOI":"10.1021\/acs.jpcb.7b09612","article-title":"Hydration and ion pairing in aqueous Mg2+ and Zn2+ solutions: Force field description aided by neutron scattering experiments and ab initio molecular dynamics simulations","volume":"122","year":"2017","journal-title":"J. Phys. Chem. B"},{"key":"2023071817355018900_c36","doi-asserted-by":"publisher","first-page":"15460","DOI":"10.1021\/acs.jpcb.5b10423","article-title":"Force field for Mg2+, Mn2+, Zn2+, and Cd2+ ions that have balanced interactions with nucleic acids","volume":"119","year":"2015","journal-title":"J. Phys. Chem. B"},{"key":"2023071817355018900_c37","doi-asserted-by":"publisher","first-page":"1011","DOI":"10.1002\/jcc.540130812","article-title":"The weighted histogram analysis method for free-energy calculations on biomolecules. I. The method","volume":"13","year":"1992","journal-title":"J. Comput. Chem."},{"key":"2023071817355018900_c38","doi-asserted-by":"publisher","first-page":"2577","DOI":"10.1002\/bip.360221211","article-title":"Dictionary of protein secondary structure: Pattern recognition of hydrogen-bonded and geometrical features","volume":"22","year":"1983","journal-title":"Biopolymers"},{"key":"2023071817355018900_c39","doi-asserted-by":"publisher","first-page":"D364","DOI":"10.1093\/nar\/gku1028","article-title":"A series of PDB-related databanks for everyday needs","volume":"43","year":"2015","journal-title":"Nucl. Acids Res."},{"key":"2023071817355018900_c40"},{"key":"2023071817355018900_c41","doi-asserted-by":"publisher","first-page":"1790","DOI":"10.1073\/pnas.0812752106","article-title":"Independent saturation of three TrpRS subsites generates a partially-assembled state similar to those observed in molecular simulations","volume":"106","year":"2009","journal-title":"Proc. Natl. Acad. Sci. U. S. A."},{"key":"2023071817355018900_c42","doi-asserted-by":"publisher","first-page":"9","DOI":"10.1016\/j.sbi.2015.11.001","article-title":"Applications of discrete molecular dynamics in biology and medicine","volume":"37","year":"2016","journal-title":"Curr. Opin. Struct. Biol."},{"key":"2023071817355018900_c43","doi-asserted-by":"publisher","first-page":"450","DOI":"10.1016\/j.tibtech.2005.07.001","article-title":"Simple but predictive protein models","volume":"23","year":"2005","journal-title":"Trends Biotechnol."},{"year":"2012","key":"2023071817355018900_c44"},{"year":"2014","key":"2023071817355018900_c45"},{"key":"2023071817355018900_c46","doi-asserted-by":"publisher","first-page":"510","DOI":"10.1002\/pro.2230","article-title":"Protein\u2013protein interactions: General trends in the relationship between binding affinity and interfacial buried surface area","volume":"22","year":"2013","journal-title":"Protein Sci."},{"year":"2007","key":"2023071817355018900_c47"},{"key":"2023071817355018900_c48","doi-asserted-by":"publisher","first-page":"1047","DOI":"10.1016\/j.str.2005.04.009","article-title":"Scaling behavior and structure of denatured proteins","volume":"13","year":"2005","journal-title":"Structure"},{"key":"2023071817355018900_c49","doi-asserted-by":"publisher","first-page":"38590","DOI":"10.1074\/jbc.M110.136911","article-title":"Tryptophanyl-tRNA synthetase urzyme: A model to recapitulate molecular evolution and investigate intramolecular complementation","volume":"285","year":"2010","journal-title":"J. Biol. Chem."},{"key":"2023071817355018900_c50","doi-asserted-by":"publisher","first-page":"1661","DOI":"10.1021\/acschembio.5b01011","article-title":"An ancestral tryptophanyl-tRNA synthetase precursor achieves high catalytic rate enhancement without ordered ground-state tertiary structures","volume":"11","year":"2016","journal-title":"ACS Chem. Biol."},{"key":"2023071817355018900_c51","doi-asserted-by":"publisher","first-page":"851","DOI":"10.1016\/j.molcel.2007.02.010","article-title":"A minimal TrpRS catalytic domain supports sense\/antisense ancestry of class I and II aminoacyl-tRNA synthetases","volume":"25","year":"2007","journal-title":"Mol. Cell"},{"key":"2023071817355018900_c52","doi-asserted-by":"publisher","first-page":"301","DOI":"10.1002\/prot.340200403","article-title":"Intrinsic secondary structure propensities of the amino acids, using statistical \u03d5-","volume":"20","year":"1994","journal-title":"Proteins"},{"key":"2023071817355018900_c53","doi-asserted-by":"publisher","first-page":"1202","DOI":"10.1038\/nsmb1325","article-title":"Structure and dynamics of a molten globular enzyme","volume":"14","year":"2007","journal-title":"Nat. Struct. Mol. Biol."},{"key":"2023071817355018900_c54","doi-asserted-by":"publisher","first-page":"12860","DOI":"10.1073\/pnas.0404109101","article-title":"An enzymatic molten globule: Efficient coupling of folding and catalysis","volume":"101","year":"2004","journal-title":"Proc. Natl. Acad. Sci. U. S. A."},{"key":"2023071817355018900_c55","first-page":"103","article-title":"Coding of class I and II aminoacyl-tRNA synthetases","year":"2017","journal-title":"Protein Reviews"}],"container-title":["Structural Dynamics"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/pubs.aip.org\/aca\/sdy\/article-pdf\/doi\/10.1063\/4.0000181\/18049544\/044301_1_4.0000181.pdf","content-type":"application\/pdf","content-version":"vor","intended-application":"text-mining"},{"URL":"https:\/\/pubs.aip.org\/aca\/sdy\/article-pdf\/doi\/10.1063\/4.0000181\/18049544\/044301_1_4.0000181.pdf","content-type":"application\/pdf","content-version":"vor","intended-application":"syndication"},{"URL":"https:\/\/pubs.aip.org\/aca\/sdy\/article-pdf\/doi\/10.1063\/4.0000181\/18049544\/044301_1_4.0000181.pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,5,9]],"date-time":"2025-05-09T17:37:26Z","timestamp":1746812246000},"score":1,"resource":{"primary":{"URL":"https:\/\/pubs.aip.org\/sdy\/article\/10\/4\/044301\/2903038\/Microcalorimetry-reveals-multi-state-thermal"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2023,7,1]]},"references-count":55,"journal-issue":{"issue":"4","published-print":{"date-parts":[[2023,7,1]]}},"URL":"https:\/\/doi.org\/10.1063\/4.0000181","relation":{},"ISSN":["2329-7778"],"issn-type":[{"type":"electronic","value":"2329-7778"}],"subject":[],"published-other":{"date-parts":[[2023,7]]},"published":{"date-parts":[[2023,7,1]]},"article-number":"044301"}}