Dimer/monomer equilibrium and domain separations of Escherichia coli ribosomal protein L7/L12.
The dimer to monomer equilibrium and interdomain separations of cysteine variants of L7/L12 have been investigated using fluorescence spectroscopy. Steady-state polarization measurements on cysteine containing variants of L7/L12, labeled with 5-(iodoacetamido)fluorescein, demonstrated dimer to monomer dissociation constants near 30 nM for variants labeled at position 33, in the N-terminal domain, and positions 63 and 89, in the C-terminal domain. A dissociation constant near 300 nM was determined for a variant labeled at position 12, in the N-terminal domain. The polarization of a labeled C-terminal fragment did not change over the range of 200 microM to 1 nM, indicating that this construct remains monomeric at these concentrations, whereas a dimer to monomer dissociation constant near 300 nM was observed for an FITC labeled N-terminal fragment. Intersubunit fluorescence resonance energy self-transfer was observed when appropriate probes were attached to cysteines at residues 12 or 33, located in the N-terminal domain. Probes attached to cysteines at positions 63 or 89 in the C-terminal domain, however, did not exhibit intersubunit self-transfer. These results indicate that these residues in the C-terminal domains are, on average, separated by greater than 85 A. Intersubunit self-transfer does occur in a C-89 double mutation variant lacking 11 residues in the putative hinge region, indicating that the loss of the hinge region brings the two C-terminal domains closer together. Rapid subunit exchange between unlabeled wild-type L7/L12 and L7/L12 variants labeled in the N-terminal domain was also demonstrated by the loss of self-transfer upon mixing of the two proteins.
Hamman
B D
BD
Department of Biochemistry and Biophysics, University of Hawaii, Honolulu 96822, USA.
Oleinikov
A V
AV
Jokhadze
G G
GG
Traut
R R
RR
Jameson
D M
DM
D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D003545:Cysteine / Q000737:chemistry; D019281:Dimerization; D004926:Escherichia coli / Q000737:chemistry* / Q000235:genetics; D019793:Fluorescein; D005452:Fluoresceins; D005454:Fluorescence Polarization; D014644:Genetic Variation; D011487:Protein Conformation; D012269:Ribosomal Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification
D001426:Bacterial Proteins; D005452:Fluoresceins; D012269:Ribosomal Proteins; C016700:ribosomal protein L7-L12; D003545:Cysteine; D019793:Fluorescein