title: Double Electron-Electron Spin Resonance Tracks Flavodoxin Folding.
abstract: Protein folding is one of the important challenges in biochemistry. Understanding the folding process requires mapping of protein structure as it folds. Here we test the potential of distance determination between paramagnetic spin-labels by a pulsed electron paramagnetic resonance method. We use double electron-electron spin resonance (DEER) to study the denaturant-dependent equilibrium folding of flavodoxin. This flavoprotein is spin-labeled with MTSL ((1-oxy-,2,2,5,5-tetramethyl-d-pyrroline-3-methyl)-methanethiosulfonate) at positions 69 and 131. We find that nativelike spin-label separation dominates the distance distributions up to 0.8 M guanidine hydrochloride. At 2.3 M denaturant, the distance distributions show an additional component, which we attribute to a folding intermediate. Upon further increase of denaturant concentration, the protein expands and evidence for a larger number of conformations than in the native state is found. We thus demonstrate that DEER is a versatile technique to expand the arsenal of methods for investigating how proteins fold.
authors.affiliation: Department of Physics, Huygens-Kamerlingh Onnes Laboratory, Leiden University , PO Box 9504, 2300 RA Leiden, The Netherlands.
authors.forename: Martin
authors.initials: M
authors.lastname: van Son
authors.affiliation: Laboratory of Biochemistry, Wageningen University , 6700 ET Wageningen, The Netherlands.
authors.forename: Simon
authors.initials: S
authors.lastname: Lindhoud
authors.affiliation: Department of Physics, Huygens-Kamerlingh Onnes Laboratory, Leiden University , PO Box 9504, 2300 RA Leiden, The Netherlands.
authors.forename: Matthijs
authors.initials: M
authors.lastname: van der Wild
authors.affiliation: Laboratory of Biochemistry, Wageningen University , 6700 ET Wageningen, The Netherlands.
authors.forename: Carlo P M
authors.initials: CP
authors.lastname: van Mierlo
authors.affiliation: Department of Physics, Huygens-Kamerlingh Onnes Laboratory, Leiden University , PO Box 9504, 2300 RA Leiden, The Netherlands.
authors.forename: Martina
authors.initials: M
authors.lastname: Huber
mesh_terms: D004578:Electron Spin Resonance Spectroscopy; D005418:Flavodoxin / Q000737:chemistry*; D008958:Models, Molecular; D017510:Protein Folding
chemical_list: D005418:Flavodoxin