title: Probing mutation-induced structural perturbations by refinement against residual dipolar couplings: application to the U4 spliceosomal RNP complex.
abstract: Confident interpretation of biochemical experiments performed with mutated proteins relies on verification of the integrity of the mutant structures. We present a simple and rapid refinement protocol for comparing the structures of mutated and wild-type proteins. Our approach involves measurement of residual dipolar couplings, and only requires assignment of the backbone resonances of the mutant species. We demonstrate application of the protocol to a mutant of the 15.5K protein, a core component of the U4 spliceosomal ribonucleoprotein (RNP) complex. Confirmation of the unperturbed structure of the mutated protein prompted re-examination of a previous mutagenesis study and indicated that the interpretation of mutant binding affinities in terms of direct interfacial contacts should be applied with caution.
authors.affiliation: Structural and Computational Biology, European Molecular Biology Laboratory, Heidelberg, Germany.
authors.forename: John P
authors.initials: JP
authors.lastname: Kirkpatrick
authors.forename: Ping
authors.initials: P
authors.lastname: Li
authors.forename: Teresa
authors.initials: T
authors.lastname: Carlomagno
mesh_terms: D001483:Base Sequence; D004586:Electrophoresis; D006801:Humans; D055029:Inverted Repeat Sequences; D008958:Models, Molecular; D016296:Mutagenesis; D050505:Mutant Proteins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D009154:Mutation; D011487:Protein Conformation; D012342:RNA, Small Nuclear / Q000235:genetics / Q000378:metabolism; D015203:Reproducibility of Results; D017414:Ribonucleoprotein, U4-U6 Small Nuclear / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism
chemical_list: D050505:Mutant Proteins; D012342:RNA, Small Nuclear; D017414:Ribonucleoprotein, U4-U6 Small Nuclear