title: Fluorescence labeling, purification, and immobilization of a double cysteine mutant calmodulin fusion protein for single-molecule experiments.
abstract: We present a method of labeling and immobilizing a low-molecular-weight protein, calmodulin (CaM), by fusion to a larger protein, maltose binding protein (MBP), for single-molecule fluorescence experiments. Immobilization in an agarose gel matrix eliminates potential interactions of the protein and the fluorophore(s) with a glass surface and allows prolonged monitoring of protein dynamics. The small size of CaM hinders its immobilization in low-weight-percentage agarose gels; however, fusion of CaM to MBP via a flexible linker provides sufficient restriction of translational mobility in 1% agarose gels. Cysteine residues were engineered into MBP.CaM (MBP-T34C,T110C-CaM) and labeled with donor and acceptor fluorescent probes yielding a construct (MBP.CaM-DA) which can be used for single-molecule single-pair fluorescence resonance energy transfer (spFRET) experiments. Mass spectrometry was used to verify the mass of MBP.CaM-DA. Assays measuring the activity of CaM reveal minimal activity differences between wild-type CaM and MBP.CaM-DA. Single-molecule fluorescence images of the donor and acceptor dyes were fit to a two-dimensional Gaussian function to demonstrate colocalization of donor and acceptor dyes. FRET is demonstrated both in bulk fluorescence spectra and in fluorescence trajectories of single MBP.CaM-DA molecules. The extension of this method to other biomolecules is also proposed.
authors.affiliation: Department of Chemistry, University of Kansas, Lawrence, KS 66045, USA.
authors.forename: Michael W
authors.initials: MW
authors.lastname: Allen
authors.forename: Ramona J Bieber
authors.initials: RJ
authors.lastname: Urbauer
authors.forename: Asma
authors.initials: A
authors.lastname: Zaidi
authors.forename: Todd D
authors.initials: TD
authors.lastname: Williams
authors.forename: Jeffrey L
authors.initials: JL
authors.lastname: Urbauer
authors.forename: Carey K
authors.initials: CK
authors.lastname: Johnson
mesh_terms: D002147:Calmodulin / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D002352:Carrier Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D003545:Cysteine / Q000737:chemistry* / Q000235:genetics; D058619:Maltose-Binding Proteins; D013058:Mass Spectrometry; D009154:Mutation; D011993:Recombinant Fusion Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D013050:Spectrometry, Fluorescence; D013388:Succinimides; D014966:Xanthenes
chemical_list: C492694:Alexa Fluor 488 carboxylic acid succinimidyl ester; D002147:Calmodulin; D002352:Carrier Proteins; D058619:Maltose-Binding Proteins; D011993:Recombinant Fusion Proteins; D013388:Succinimides; D014966:Xanthenes; C034657:Texas red; D003545:Cysteine