Comparative cross-linking study on the 50S ribosomal subunit from Escherichia coli

Abstract

We have carried out an extensive protein-protein cross-linking study on the 50S ribosomal subunit of Escherichia coli using four different cross-linking reagents of varying length and specificity. For the unambiguous identification of the members of the cross-linked protein complexes, immunoblotting techniques using antisera specific for each individual ribosomal protein have been used, and for each cross-link, the cross-linking yield has been determined. With the smallest cross-linking reagent diepoxybutane (4 A), four cross-links have been identified, namely, L3-L19, L10-L11, L13-L21, and L14-L19. With the sulfhydryl-specific cross-linking reagent o-phenylenedimaleimide (5.2 A) and p-phenylenedimaleimide (12 A), the cross-links L2-L9, L3-L13, L3-L19, L9-L28, L13-L20, L14-L19, L16-L27, L17-L32, and L20-L21 were formed; in addition, the cross-link L23-L29 was exclusively found with the shorter o-phenylenedimaleimide. The cross-links obtained with dithiobis(succinimidyl propionate) (12 A) were L1-L33, L2-L9, L2-L9-L28, L3-L19, L9-L28, L13-L21, L14-L19, L16-L27, L17-L32, L19-L25, L20-L21, and L23-L34. The good agreement of the cross-links obtained with the different cross-linking reagents used in this study demonstrates the reliability of our cross-linking approach. Incorporation of our cross-linking results into the three-dimensional model of the 50S ribosomal subunit derived from immunoelectron microscopy yields the locations for 29 of the 33 proteins within the larger ribosomal subunit.

The GTP-binding Protein YlqF Participates in the Late Step of 50 S Ribosomal Subunit Assembly in Bacillus subtilis

“…Both L16 and L27 proteins belong to the late assembly protein (37,39,40). Furthermore, L16 cross-links with L27 in the mature 50 S subunit (41,42). Incomplete assembly of the 50 S subunit without L16 leads to defects in peptidyl transferase activity (43,44), peptidyl-tRNA hydrolysis activity (45), association with the 30 S subunit (46), and binding of aminoacyl-tRNA (47).…”

HybF, a Zinc-Containing Protein Involved in NiFe Hydrogenase Maturation

“…This was done as described by Mehta et al (33) by using dimethyl suberimidate, a homobifunctional imidoester cross-linker specific for primary amines (57). Alternatively, the sulfhydryl-specific cross-linking reagent p-phenylenedimaleimide was used (56).…”

Subunit arrangement in the human 20S proteasome

“…Crosslinking was performed with cleavable crosslinkers as described (25)(26)(27). Purified proteasomes (500 g) were mixed with 100 M (final concentration) dithiobis(succinimidylpropionate) (DSP; Pierce) in a final volume of 1.25 ml 5 mM N-tris [hydroxymethyl]methyl-3-aminopropanesulfonic acid (TAPS; Sigma).…”