title: Förster resonance energy transfer studies of calmodulin produced by native protein ligation reveal inter-domain electrostatic repulsion.
abstract: This study explores the influence of long-range intra-protein electrostatic interactions on the conformation of calmodulin in solution. Ensemble Förster resonance energy transfer (FRET) is measured for calmodulin with a fluorophore pair incorporated specifically with a donor at residue 17 and an acceptor at position 117. This construct was generated by a combination of solid phase peptide synthesis, cloning, expression and native chemical ligation. This labelling method has not previously been used with calmodulin and represents a convenient method for ensuring the explicit positioning of the fluorophores. The ensemble FRET experiments reveal significant electrostatic repulsion between the globular domains in the calcium-free protein. At low salt, calmodulin has a relatively extended conformation and the distance between the domains is further increased by denaturation, by heat or by non-ionic denaturants. The repulsion between domains is screened by salt and is also diminished by calcium binding, which changes the protein net charge from -23 to -15. Compared with the calcium-free form at low salt, the FRET efficiency for the calcium-bound form has, on average, increased 10-fold. The conformation of the calcium form is insensitive to salt screening. These results imply that when the two globular domains of calmodulin interact with target, there is no significant free energy penalty due to electrostatic interactions.
authors.affiliation: Biophysical Chemistry, Chemical Centre, Lund University, SE-221 00 Lund, Sweden.
authors.forename: Erik
authors.initials: E
authors.lastname: Hellstrand
authors.forename: Stephanie
authors.initials: S
authors.lastname: Kukora
authors.forename: Cynthia F
authors.initials: CF
authors.lastname: Shuman
authors.forename: Sara
authors.initials: S
authors.lastname: Steenbergen
authors.forename: Eva
authors.initials: E
authors.lastname: Thulin
authors.forename: Anita
authors.initials: A
authors.lastname: Kohli
authors.forename: Beth
authors.initials: B
authors.lastname: Krouse
authors.forename: Sara
authors.initials: S
authors.lastname: Linse
authors.forename: Karin S
authors.initials: KS
authors.lastname: Åkerfeldt
mesh_terms: D001665:Binding Sites; D002118:Calcium / Q000737:chemistry / Q000378:metabolism; D020013:Calcium Signaling; D002147:Calmodulin / Q000737:chemistry* / Q000378:metabolism; D031541:Fluorescence Resonance Energy Transfer; D005456:Fluorescent Dyes / Q000737:chemistry; D006801:Humans; D008301:Maleimides / Q000737:chemistry; D008958:Models, Molecular; D009994:Osmolar Concentration; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D011487:Protein Conformation; D011489:Protein Denaturation; D017434:Protein Structure, Tertiary; D011806:Quinolinium Compounds / Q000737:chemistry; D055672:Static Electricity
chemical_list: C504424:Alexa Fluor 488 C5-maleimide; C481052:Alexa fluor 546; D002147:Calmodulin; D005456:Fluorescent Dyes; D008301:Maleimides; D010446:Peptide Fragments; D011806:Quinolinium Compounds; D002118:Calcium