title: Rotational and conformational dynamics of Escherichia coli ribosomal protein L7/L12.
abstract: Fluorescence methods were utilized to study dynamic aspects of the 24 kDa dimeric Escherichia coli ribosomal protein L7/L12. Oligonucleotide site-directed mutagenesis was used to introduce cysteine residues at specific locations along the peptide chain, in both the C-terminal and N-terminal domains, and various sulfhydryl reactive fluorescence probes (iodoacetamido) fluorescein, IAEDANS, pyrenemethyl iodoacetate) were attached to these residues. In addition to the full-length proteins, a hinge-deleted variant and variants corresponding to the C-terminal fragment and the N-terminal fragment were also studied. Both steady-state and time-resolved fluorescence measurements were carried out, and the results demonstrated that L7/L12 is not a rigid molecule. Specifically, the two C-terminal domains move freely with respect to one another and with respect to the dimeric N-terminal domain. Removal of the hinge region, however, significantly reduces the mobility of the C-terminal domains. The data also show that the rotational relaxation time monitored by the fluorescent probe-depends upon the probe's excited state lifetime. This observation is interpreted to indicate that a hierarchy of motions exists in the L7/L12 molecule including facile motions of the C-terminal domains and dimeric N-terminal domain, in addition to the overall tumbling of the protein. Probes attached to the N-terminal domain exhibit global rotational relaxation times consistent with the molecular mass of the dimeric N-terminal fragment. Upon reconstitution of labeled L7/L12 with ribosomal cores, however, the motion associated with the dimeric N-terminal domain is greatly diminished while the facile motion of the C-terminal domains is almost unchanged.
authors.affiliation: Department of Biochemistry and Biophysics, University of Hawaii, Honolulu 96822, USA.
authors.forename: B D
authors.initials: BD
authors.lastname: Hamman
authors.forename: A V
authors.initials: AV
authors.lastname: Oleinikov
authors.forename: G G
authors.initials: GG
authors.lastname: Jokhadze
authors.forename: R R
authors.initials: RR
authors.lastname: Traut
authors.forename: D M
authors.initials: DM
authors.lastname: Jameson
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics; D019281:Dimerization; D004926:Escherichia coli / Q000737:chemistry / Q000235:genetics; D005454:Fluorescence Polarization; D005456:Fluorescent Dyes; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics; D012269:Ribosomal Proteins / Q000737:chemistry* / Q000235:genetics; D013816:Thermodynamics
chemical_list: D001426:Bacterial Proteins; D005456:Fluorescent Dyes; D011994:Recombinant Proteins; D012269:Ribosomal Proteins; C016700:ribosomal protein L7-L12