title: Conformational changes of 30S ribosomes measured by intrinsic and extrinsic fluorescence.
abstract: The intrinsic tryptophan fluorescence and the fluorescence of N-(3-pyrene)maleimide, a covalently bound sulfhydryl-specific extrinsic probe, have been used to study the conformation of the 30S ribosomal subunit of Escherichia coli. (a) The tryptophan fluorescenct spectrum of the free ribosomal proteins is shifted to shorter wavelengths than that of free tryptophan. When the proteins are incorporated into the organized structure of the ribosome, there is a small additional blue shift and the emission band becomes narrower. In 6 M urea, the spectrum of the proteins, whether free or in the ribosome, becomes identical with that of the amino acid, reflecting exposure of previously shielded tryptophan residues. (b) When magnesium-depleted ribosomes are unfolded at low ionic strength, the tryptophan fluorescence spectrum changes, although circular dichroism shows no change in alpha-helix content of the proteins. (c) Intrinsic and extrinsic fluorescence were both found to be sensitive to a limited and fully reversible transition that takes place when ribosomes are incubated under conditions that increase their activity in vitro. This suggests that both probes may be of use in monitoring conformational changes that occur under conditions consistent with activity. The kinetics of the concurrent changes in extrinsic fluorescence and aminoacyl-tRNA binding activity were compared. (d) Conditions are described for labeling ribosomes with N-(3-pyrene)maleimide without impairing their activity.
mesh_terms: D002942:Circular Dichroism; D004926:Escherichia coli; D005453:Fluorescence; D008274:Magnesium / Q000494:pharmacology; D008301:Maleimides; D011487:Protein Conformation / Q000187:drug effects; D011721:Pyrenes; D012269:Ribosomal Proteins; D012270:Ribosomes; D014364:Tryptophan; D014508:Urea / Q000494:pharmacology
chemical_list: D008301:Maleimides; D011721:Pyrenes; D012269:Ribosomal Proteins; D014364:Tryptophan; D014508:Urea; D008274:Magnesium