Open AccessDistance measurement by energy transfer
Author(s) -
STEINHÄUSER Klaus G.,
WOOLLEY Paul,
DIJK Jan,
EPE Bernd
Publication year - 1983
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1111/j.1432-1033.1983.tb07834.x
Subject(s) - ribonucleoprotein , 50s , fluorophore , protein subunit , chemistry , ribosomal rna , ribosomal protein , biophysics , thiol , fluorescence , crystallography , stereochemistry , rna , transfer rna , biochemistry , biology , physics , ribosome , optics , gene
Ribosomal proteins L6, L11 and the complex [(L12) 4 · L10] were labelled specifically at their respective single thiol groups, either with the acetylaminoethyl‐dansyl or with the acetamidofluorescein fluorophore. The labelled proteins were then reconstituted, singly or in pairs, into ribosomal 50 S subunits; the presence of the label had no observable effect on the composition, shape or activity of the reconstituted subunits. The distances between the labelled thiol groups were measured by a fluorescence energy transfer method detailed elsewhere [Epe, B. et al. (1983) Proc. Natl Acad. Sci. USA, 80 , 2579–2583] and were found to be: for L6–L10, 60 Å (6.0 nm); for L6–L11, 46 Å (4.6 nm); for L10–L11, 56 Å (5.6 nm). Reversal of the direction of energy transfer by exchanging labels gave duplicate distances which differed, on average, by about 4%. The distance between the fluorescent labels on L10 and L11 in the [23 S‐RNA · L10 · L11 · (L12) 4 ] ribonucleoprotein complex was the same as in the 50S subunit, but all three distances were greater in 50S subunits which had been reconstituted without the final activation step (incubation at 50°C). This suggests a tightening of the L6/L10/L11 domain of the 50S subunit during the activation step.