title: Assembly states of the nucleosome assembly protein 1 (NAP-1) revealed by sedimentation velocity and non-denaturing MS.
abstract: Proteins often exist as ensembles of interconverting states in solution which are often difficult to quantify. In the present manuscript we show that the combination of MS under nondenaturing conditions and AUC-SV (analytical ultracentrifugation sedimentation velocity) unambiguously clarifies a distribution of states and hydrodynamic shapes of assembled oligomers for the NAP-1 (nucleosome assembly protein 1). MS established the number of associated units, which was utilized as input for the numerical analysis of AUC-SV profiles. The AUC-SV analysis revealed that less than 1% of NAP-1 monomer exists at the micromolar concentration range and that the basic assembly unit consists of dimers of yeast or human NAP-1. These dimers interact non-covalently to form even-numbered higher-assembly states, such as tetramers, hexamers, octamers and decamers. MS and AUC-SV consistently showed that the formation of the higher oligomers was suppressed with increasing ionic strength, implicating electrostatic interactions in the formation of higher oligomers. The hydrodynamic shapes of the NAP-1 tetramer estimated from AUC-SV agreed with the previously proposed assembly models built using the known three-dimensional structure of yeast NAP-1. Those of the hexamer and octamer could be represented by new models shown in the present study. Additionally, MS was used to measure the stoichiometry of the interaction between the human NAP-1 dimer and the histone H2A-H2B dimer or H3-H4 tetramer. The present study illustrates a rigorous procedure for the analysis of protein assembly and protein-protein interactions in solution.
authors.affiliation: Graduate School of Engineering, Osaka University, Suita 565-0871, Japan.
authors.forename: Masanori
authors.initials: M
authors.lastname: Noda
authors.forename: Susumu
authors.initials: S
authors.lastname: Uchiyama
authors.forename: Adam R
authors.initials: AR
authors.lastname: McKay
authors.forename: Akihiro
authors.initials: A
authors.lastname: Morimoto
authors.forename: Shigeki
authors.initials: S
authors.lastname: Misawa
authors.forename: Akihiro
authors.initials: A
authors.lastname: Yoshida
authors.forename: Hideto
authors.initials: H
authors.lastname: Shimahara
authors.forename: Hiroto
authors.initials: H
authors.lastname: Takinowaki
authors.forename: Shota
authors.initials: S
authors.lastname: Nakamura
authors.forename: Yuji
authors.initials: Y
authors.lastname: Kobayashi
authors.forename: Sachihiro
authors.initials: S
authors.lastname: Matsunaga
authors.forename: Tadayasu
authors.initials: T
authors.lastname: Ohkubo
authors.forename: Carol V
authors.initials: CV
authors.lastname: Robinson
authors.forename: Kiichi
authors.initials: K
authors.lastname: Fukui
mesh_terms: D019281:Dimerization; D006657:Histones / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D013058:Mass Spectrometry / Q000379:methods*; D056490:Nucleosome Assembly Protein 1 / Q000737:chemistry* / Q000378:metabolism; D012996:Solutions / Q000737:chemistry / Q000378:metabolism; D014461:Ultracentrifugation / Q000379:methods*
chemical_list: D006657:Histones; D056490:Nucleosome Assembly Protein 1; D012996:Solutions