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• W2072571320 abstract "We used single-pair fluorescence resonance energy transfer (spFRET) measurements to characterize denatured and partially denatured states of the multidomain calcium signaling protein calmodulin (CaM) in both its apo and Ca2+-bound forms. The results demonstrate the existence of an unfolding intermediate. A CaM mutant (CaM-T34C-T110C) was doubly labeled with fluorescent probes AlexaFlour 488 and Texas Red at opposing globular domains. Single-molecule distributions of the distance between fluorophores were obtained by spFRET at varying levels of the denaturant urea. Multiple conformational states of CaM were observed, and the amplitude of each conformation was dependent on urea concentration, with the amplitude of an extended conformation increasing upon denaturation. The distributions at intermediate urea concentrations could not be adequately described as a combination of native and denatured conformations, showing that CaM does not denature via a two-state process and demonstrating that at least one intermediate is present. The intermediate conformations formed upon addition of urea were different for Ca2+-CaM and apoCaM. An increase in the amplitude of a compact conformation in CaM was observed for apoCaM but not for Ca2+-CAM upon the addition of urea. The changes in the single-molecule distributions of CaM upon denaturation can be described by either a range of intermediate structures or by the presence of a single unfolding intermediate that grows in amplitude upon denaturation. A model for stepwise unfolding of CaM is suggested in which the domains of CaM unfold sequentially." @default.
• W2072571320 created "2016-06-24" @default.
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• W2072571320 date "2005-08-01" @default.
• W2072571320 modified "2023-10-16" @default.
• W2072571320 title "Sampling Unfolding Intermediates in Calmodulin by Single-Molecule Spectroscopy" @default.
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• W2072571320 doi "https://doi.org/10.1021/ja0526315" @default.
• W2072571320 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/16117552" @default.
• W2072571320 hasPublicationYear "2005" @default.
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