Tetramethylrhodamine dimer formation as a spectroscopic probe of the conformation of Escherichia coli ribosomal protein L7/L12 dimers.
The fluorescent probe tetramethylrhodamine iodoacetamide was attached to cysteine residues substituted at various specific locations in full-length and deletion variants of the homodimeric Escherichia coli ribosomal protein L7/L12. Ground-state tetramethylrhodamine dimers form between the two subunits of L7/L12 depending upon the location of the probe. The formation of tetramethylrhodamine dimers caused the appearance of a new absorption band at 518 nm that was used to estimate the extent of interaction of the probes in the different protein variants. Intersubunit tetramethylrhodamine dimers form when tetramethylrhodamine acetamide is attached to two different sites in the N-terminal domain of the L7/L12 dimer (residues 12 or 33), but not when attached to sites in the C-terminal domain (residues 63, 89, or 99). The tetramethylrhodamine dimers do form at sites in the C-terminal domain in L7/L12 variants that contain deletions of 11 or 18 residues within the putative flexible hinge that separates the N- and C-terminal domains. The tetramethylrhodamine dimers disappear rapidly (within 5 s) upon addition of excess unlabeled wild-type L7/L12. It appears that singly labeled L7/L12 dimers are formed by exchange with wild-type dimers. Binding of L7/L12:tetramethylrhodamine cysteine 33 or cysteine 12 dimers either to L7/L12-depleted ribosomal core particles, or to ribosomal protein L10 alone, results in disappearance of the 518-nm absorption band. This result implies a conformational change in the N-terminal domain of L7/L12 upon its binding to the ribosome, or to L10.
Hamman
B D
BD
Department of Biochemistry and Biophysics, University of Hawaii, Honolulu, 96822, USA.
Oleinikov
A V
AV
Jokhadze
G G
GG
Bochkariov
D E
DE
Traut
R R
RR
Jameson
D M
DM
D001665:Binding Sites; D003545:Cysteine; D004926:Escherichia coli / Q000378:metabolism*; D005456:Fluorescent Dyes; D014644:Genetic Variation; D046911:Macromolecular Substances; D008961:Models, Structural; D011487:Protein Conformation; D012235:Rhodamines; D000080882:Ribosomal Protein L10; D012269:Ribosomal Proteins / Q000737:chemistry*; D012270:Ribosomes / Q000378:metabolism; D017384:Sequence Deletion
D005456:Fluorescent Dyes; D046911:Macromolecular Substances; D012235:Rhodamines; D012269:Ribosomal Proteins; C016700:ribosomal protein L7-L12; C034996:tetramethylrhodamine iodoacetamide; D003545:Cysteine