title: Dap-SL: a new site-directed nitroxide spin labeling approach for determining structure and motions in synthesized peptides and proteins.
abstract: A new approach for site-directed placement of nitroxide spin labels in chemically synthesized peptides and proteins is described. The scheme takes advantage of a novel diaminopropionic acid scaffold to independently control backbone and side chain elongation. The result is a spin-labeled side chain, referred to as Dap-SL, in which an amide bond forms a linker between the nitroxide and the peptide backbone. The method was demonstrated in a series of helical peptides. Circular dichroism and nuclear magnetic resonance showed that Dap-SL introduces only a minor perturbation in the helical structure. The electron paramagnetic resonance spectrum of the singly labeled species allowed for determination of the spin label rotational correlation time and suggests that the Dap-SL side chain is more flexible than the modified Cys side chain frequently used in site-directed spin label studies. Spectra of the doubly labeled peptides indicate a mixture of 3(10)-helix and alpha-helix, which parallels findings from previous studies. The scheme demonstrated here offers a fundamentally new approach for introducing spin labels into proteins and promises to significantly extend biophysical investigations of large proteins and receptors. In addition, the technique is readily modified for incorporation of any biophysical probe.
authors.affiliation: Department of Chemistry and Biochemistry, University of California, Santa Cruz, CA 95064, USA.
authors.forename: Joe C
authors.initials: JC
authors.lastname: McNulty
authors.forename: Darren A
authors.initials: DA
authors.lastname: Thompson
authors.forename: Michael R
authors.initials: MR
authors.lastname: Carrasco
authors.forename: Glenn L
authors.initials: GL
authors.lastname: Millhauser
mesh_terms: D004578:Electron Spin Resonance Spectroscopy; D009589:Nitrogen Oxides / Q000737:chemistry*; D010455:Peptides / Q000737:chemistry*; D011506:Proteins / Q000737:chemistry*; D013113:Spin Labels
chemical_list: D009589:Nitrogen Oxides; D010455:Peptides; D011506:Proteins; D013113:Spin Labels