PMID:8988003
| Citation |
Hamman, BD, Oleinikov, AV, Jokhadze, GG, Traut, RR and Jameson, DM (1996) Rotational and conformational dynamics of Escherichia coli ribosomal protein L7/L12. Biochemistry 35:16672-9 |
|---|---|
| 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. |
| Links |
PubMed Online version:10.1021/bi9615001 |
| Keywords |
Bacterial Proteins/chemistry; Bacterial Proteins/genetics; Dimerization; Escherichia coli/chemistry; Escherichia coli/genetics; Fluorescence Polarization; Fluorescent Dyes; Mutagenesis, Site-Directed; Protein Conformation; Recombinant Proteins/chemistry; Recombinant Proteins/genetics; Ribosomal Proteins/chemistry; Ribosomal Proteins/genetics; Thermodynamics |
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