title: NMR analysis of methyl groups at 100-500 kDa: model systems and Arp2/3 complex.
abstract: Large macromolecular machines are among the most important and challenging targets for structural and mechanistic analyses. Consequently, there is great interest in development of NMR methods for the study of multicomponent systems in the 50-500 kDa range. Biochemical methods also must be developed in concert to produce such systems in selectively labeled form. Here, we present (1)H/(13)C-HSQC spectra of protonated methyl groups in a model system that mimics molecular weights up to approximately 560 kDa. Signals from side chain methyl groups of Ile, Leu, and Val residues are clearly detectable at correlation times up to approximately 330 ns. We have also developed a biochemical procedure to produce the 240 kDa, heteroheptameric Arp2/3 actin nucleation complex selectively labeled at one subunit and obtained (1)H/(13)C-HSQC spectra of this assembly. Sensitivity in spectra of both the Arp2/3 complex and the model system indicate that methyl groups will be useful sources of information in nonsymmetric systems with molecular weights greater than 600 kDa at concentrations less than 100 microM. Methyl analyses will complement TROSY and CRINEPT analyses of amides in NMR studies of structure and molecular interactions of extremely large macromolecules and assemblies.
authors.affiliation: Department of Biochemistry, University of Texas Southwestern Medical Center at Dallas, 5323 Harry Hines Boulevard, Dallas, Texas 75390, USA.
authors.forename: Mara
authors.initials: M
authors.lastname: Kreishman-Deitrick
authors.forename: Coumaran
authors.initials: C
authors.lastname: Egile
authors.forename: David W
authors.initials: DW
authors.lastname: Hoyt
authors.forename: Joseph J
authors.initials: JJ
authors.lastname: Ford
authors.forename: Rong
authors.initials: R
authors.lastname: Li
authors.forename: Michael K
authors.initials: MK
authors.lastname: Rosen
mesh_terms: D051377:Actin-Related Protein 2; D051378:Actin-Related Protein 3; D000199:Actins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites; D007074:Immunoglobulin G / Q000378:metabolism; D046911:Macromolecular Substances; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D008745:Methylation; D008954:Models, Biological; D008970:Molecular Weight; D011487:Protein Conformation; D021122:Protein Subunits / Q000737:chemistry / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: C477632:ARP2 protein, S cerevisiae; C102245:ARP3 protein, S cerevisiae; D051377:Actin-Related Protein 2; D051378:Actin-Related Protein 3; D000199:Actins; D007074:Immunoglobulin G; D046911:Macromolecular Substances; D021122:Protein Subunits; D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins