title: Global and local dynamics of the human U1A protein determined by tryptophan fluorescence.
abstract: Tryptophan residues have been introduced into two domains of the human U1A protein to probe solution dynamics. The full length protein contains 282 residues, separated into three distinct domains: the N-terminal RBD1 (RNA Binding Domain I), consisting of amino acids 1-101; the C-terminal RBD2, residues 202-282; and the intervening linker region. Tryptophan residues have been substituted for specific phenylalanine residues on the surface of the beta-sheet of either RBD1 or RBD2, thus introducing a single solvent exposed tryptophan as a fluorescence reporter. Both steady-state and time-resolved fluorescence measurements of the isolated RBD domains show that each tryptophan experiences a unique environment on the beta-sheet surface. The spectral properties of each tryptophan in RBD1 and RBD2 are preserved in the context of the U1A protein, indicating these domains do not interact with each other or with the linker region. The rotational correlation times of the isolated RBDs and the whole U1A, determined by dynamic polarization measurements, show that the linker region is highly flexible such that each RBD exhibits uncorrelated motion.
authors.affiliation: Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St. Louis, Missouri 63110, USA.
authors.forename: J M
authors.initials: JM
authors.lastname: Jean
authors.forename: C
authors.initials: C
authors.lastname: Clerte
authors.forename: K B
authors.initials: KB
authors.lastname: Hall
mesh_terms: D002942:Circular Dichroism; D005454:Fluorescence Polarization; D006801:Humans; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D016601:RNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics; D017412:Ribonucleoprotein, U1 Small Nuclear / Q000737:chemistry* / Q000235:genetics; D014364:Tryptophan / Q000737:chemistry*
chemical_list: D016601:RNA-Binding Proteins; D017412:Ribonucleoprotein, U1 Small Nuclear; C075339:U1A protein; D014364:Tryptophan