title: Deuterium Labeling Together with Contrast Variation Small-Angle Neutron Scattering Suggests How Skp Captures and Releases Unfolded Outer Membrane Proteins.
abstract: In Gram-negative bacteria, the chaperone protein Skp forms specific and stable complexes with membrane proteins while they are transported across the periplasm to the outer membrane. The jellyfish-like architecture of Skp is similar to the eukaryotic and archaeal prefoldins and the mitochondrial Tim chaperones, that is the α-helical "tentacles" extend from a β-strand "body" to create an internal cavity. Contrast variation small-angle neutron scattering (SANS) experiments on Skp alone in solution and bound in two different complexes to unfolded outer membrane proteins (uOMPs), OmpA and OmpW, demonstrate that the helical tentacles of Skp bind their substrate in a clamp-like mechanism in a conformation similar to that previously observed in the apo crystal structure of Skp. Deuteration of the uOMP component combined with contrast variation analysis allowed the shapes of Skp and uOMP as well as the location of uOMP with respect to Skp to be determined in both complexes. This represents unique information that could not be obtained without deuterium labeling of the uOMPs. The data yield the first direct structural evidence that the α-helical Skp tentacles move closer together on binding its substrate and that the structure of Skp is different when binding different uOMPs. This work presents, by example, a tutorial on performing SANS experiments using both deuterium labeling and contrast variation, including SANS theory, sample preparation, data collection, sample quality validation, data analysis, and structure modeling.
authors.affiliation: T.C. Jenkins Department of Biophysics, Johns Hopkins University, Baltimore, Maryland, USA.
authors.forename: Nathan R
authors.initials: NR
authors.lastname: Zaccai
authors.affiliation: T.C. Jenkins Department of Biophysics, Johns Hopkins University, Baltimore, Maryland, USA.
authors.forename: Clifford W
authors.initials: CW
authors.lastname: Sandlin
authors.affiliation: Institute for Bioscience and Biotechnology Research, University of Maryland, Rockville, Maryland, USA.
authors.forename: James T
authors.initials: JT
authors.lastname: Hoopes
authors.affiliation: National Institute of Standards and Technology, Gaithersburg, Maryland, USA.
authors.forename: Joseph E
authors.initials: JE
authors.lastname: Curtis
authors.affiliation: T.C. Jenkins Department of Biophysics, Johns Hopkins University, Baltimore, Maryland, USA.
authors.forename: Patrick J
authors.initials: PJ
authors.lastname: Fleming
authors.affiliation: T.C. Jenkins Department of Biophysics, Johns Hopkins University, Baltimore, Maryland, USA.
authors.forename: Karen G
authors.initials: KG
authors.lastname: Fleming
authors.affiliation: National Institute of Standards and Technology, Gaithersburg, Maryland, USA. Electronic address: nrz20@cam.ac.uk.
authors.forename: Susan
authors.initials: S
authors.lastname: Krueger
mesh_terms: D001425:Bacterial Outer Membrane Proteins / Q000737:chemistry*; D003287:Contrast Media / Q000737:chemistry; D003461:Crystallography; D003903:Deuterium / Q000737:chemistry*; D006801:Humans; D009502:Neutrons; D019897:Periplasm / Q000737:chemistry; D044786:S-Phase Kinase-Associated Proteins / Q000737:chemistry*; D053838:Scattering, Small Angle; D012996:Solutions / Q000737:chemistry; D013194:Staining and Labeling / Q000379:methods*; D014961:X-Ray Diffraction
chemical_list: D001425:Bacterial Outer Membrane Proteins; D003287:Contrast Media; D044786:S-Phase Kinase-Associated Proteins; D012996:Solutions; D003903:Deuterium
keywords: Disordered protein; Holdase; Jellyfish-like chaperone; Outer membrane protein (OMP) transport; Periplasm; SANS; Skp; Small-angle neutron scattering