NMR characterisation of the relationship between frustration and the excited state of Im7 |
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Authors: | Whittaker Sara B-M Clayden Nigel J Moore Geoffrey R |
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Institution: | 1 Centre for Structural and Molecular Biochemistry, School of Chemistry, University of East Anglia, Norwich NR4 7TJ, UK2 The Henry Wellcome Building for Biomolecular NMR Spectroscopy, School of Cancer Sciences, University of Birmingham, Vincent Drive, Edgbaston, Birmingham B15 2TT, UK |
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Abstract: | Previous work shows that Im9 folds in a two-state transition while its homologue Im7 folds in a three-state transition via an on-pathway kinetic intermediate state (KIS), with this difference being related to frustration in the structure of Im7. We have used NMR spectroscopy to study conformational dynamics connected to the frustration. A combination of equilibrium peptide N1H/N2H exchange, model-free analyses of backbone NH relaxation data and relaxation dispersion (RD)-NMR shows that the native state of Im7 is in equilibrium with an intermediate state that is lowly populated equilibrium intermediate state (EIS)]. Comparison of kinetic and thermodynamic parameters describing the EIS native-state equilibrium obtained by RD-NMR with previously reported parameters describing the KIS native-state equilibrium obtained from stopped-flow fluorescence studies of refolding His-tagged Im7 shows that the KIS and the EIS are the same species. 15N chemical shifts of the EIS obtained from the RD-NMR analysis show that residues forming helix III in the native state are unstructured in the EIS while other residues experiencing frustration in the native state are in structured regions of the EIS. We show that binding of Im7 and its L53A/I54A variant (which resembles the EIS as shown in previous work) to the cognate partner for Im7, the DNase domain of colicin E7, causes the dynamic processes associated with the frustration to be dampened. |
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Keywords: | CPMG Carr-Purcell-Meiboom-Gill   EIS equilibrium intermediate state HSQC heteronuclear single quantum coherence Im2 Im7 and Im9 the immunity proteins for colicins E2 E7 and E9 respectively Im7? His-tagged Im7 KIS kinetic intermediate state MD molecular dynamics NOE nuclear Overhauser enhancement PDB Protein Data Bank RD relaxation dispersion |
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