Sampling unfolding intermediates in calmodulin by single-molecule spectroscopy.
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 Ca(2+)-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 Ca(2+)-CaM and apoCaM. An increase in the amplitude of a compact conformation in CaM was observed for apoCaM but not for Ca(2+)-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.
Slaughter
Brian D
BD
Department of Chemistry, 1251 Wescoe Hall Drive, University of Kansas, Lawrence, Kansas 66045-7582, USA.
Unruh
Jay R
JR
Price
E Shane
ES
Huynh
Jason L
JL
Bieber Urbauer
Ramona J
RJ
Johnson
Carey K
CK
D002118:Calcium / Q000737:chemistry / Q000378:metabolism; D002147:Calmodulin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D009154:Mutation; D011485:Protein Binding; D011487:Protein Conformation; D011489:Protein Denaturation; D014508:Urea / Q000737:chemistry*
D002147:Calmodulin; D014508:Urea; D002118:Calcium