George S M Hanson, Faidra Batsaki, Teagan L Myerscough, Kristin Piché, Ariel Louwrier, Christopher R Coxon
{"title":"<sup>19</sup>F NMR-tags for peptidyl prolyl conformation analysis.","authors":"George S M Hanson, Faidra Batsaki, Teagan L Myerscough, Kristin Piché, Ariel Louwrier, Christopher R Coxon","doi":"10.1039/d5cb00118h","DOIUrl":null,"url":null,"abstract":"<p><p>Proline <i>cis</i>/<i>trans</i> isomerism plays an important role in protein folding and mediating protein-protein interactions in short linear interacting motifs within intrinsically disordered protein regions. The slow exchange rate between <i>cis</i> and <i>trans</i> prolyl bonds provides distinct signals in <sup>19</sup>F NMR analysis of fluorinated peptides, allowing for simple quantification of each population. However, fluorine is not naturally found in proteins but can be introduced using chemical tags. In this study, we evaluate a range of fluorinated cysteine-reactive <sup>19</sup>F NMR tags to assess their ability to react with short, linear proline-containing peptides and accurately report on the equilibrium <i>cis</i>/<i>trans</i>-Pro populations. Several fluorinated electrophilic tags, including nitrobenzenes, sulfonylpyrimidines, and acrylamides, were found to react chemoselectively and reliably report on the %<i>cis</i>-Pro in the model peptide Ac-LPAAC. Other <sup>19</sup>F NMR tags were found to be poor reporters of local proline conformation. Although pentafluoropyridine was non-chemoselective, it still reliably reported on %<i>cis</i>-Pro when conjugated <i>via</i> cysteine or tyrosine in Ac-LPAAX (X = Cys, Tyr, Lys) peptides. 3,4-Difluoronitrobenzene was found to be compatible with protein tagging, albeit it had modest reactivity and afforded a pair of regioisimeric tagging-products when reacted with a cysteine mutant of α-synuclein. These tools may be valuable for probing <i>cis</i>/<i>trans</i>-Pro populations in proteins.</p>","PeriodicalId":40691,"journal":{"name":"RSC Chemical Biology","volume":" ","pages":""},"PeriodicalIF":3.1000,"publicationDate":"2025-09-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12435502/pdf/","citationCount":"0","resultStr":null,"platform":"Semanticscholar","paperid":null,"PeriodicalName":"RSC Chemical Biology","FirstCategoryId":"1085","ListUrlMain":"https://doi.org/10.1039/d5cb00118h","RegionNum":0,"RegionCategory":null,"ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q2","JCRName":"BIOCHEMISTRY & MOLECULAR BIOLOGY","Score":null,"Total":0}
引用次数: 0
Abstract
Proline cis/trans isomerism plays an important role in protein folding and mediating protein-protein interactions in short linear interacting motifs within intrinsically disordered protein regions. The slow exchange rate between cis and trans prolyl bonds provides distinct signals in 19F NMR analysis of fluorinated peptides, allowing for simple quantification of each population. However, fluorine is not naturally found in proteins but can be introduced using chemical tags. In this study, we evaluate a range of fluorinated cysteine-reactive 19F NMR tags to assess their ability to react with short, linear proline-containing peptides and accurately report on the equilibrium cis/trans-Pro populations. Several fluorinated electrophilic tags, including nitrobenzenes, sulfonylpyrimidines, and acrylamides, were found to react chemoselectively and reliably report on the %cis-Pro in the model peptide Ac-LPAAC. Other 19F NMR tags were found to be poor reporters of local proline conformation. Although pentafluoropyridine was non-chemoselective, it still reliably reported on %cis-Pro when conjugated via cysteine or tyrosine in Ac-LPAAX (X = Cys, Tyr, Lys) peptides. 3,4-Difluoronitrobenzene was found to be compatible with protein tagging, albeit it had modest reactivity and afforded a pair of regioisimeric tagging-products when reacted with a cysteine mutant of α-synuclein. These tools may be valuable for probing cis/trans-Pro populations in proteins.