Location and domain structure of Escherichia coli ribosomal protein L7/L12: site specific cysteine crosslinking and attachment of fluorescent probes.
Five different variants of L7/L12 containing single cysteine substitutions, two in the N-terminal (NTD) and three in the C-terminal domain (CTD), were produced, modified with [125I]N-[4-(p-azidosalicylamido)butyl]-3-(2'-pyridyldithio) propionamide ([125I]APDP), a sulfhydryl-specific, heterobifunctional, cleavable photo-cross-linking reagent, and reconstituted into ribosomes. These were irradiated, the total proteins were extracted and reductively cleaved, and the cross-linked proteins were identified. The effect of zero-length disulfide cross-linking on binding and activity was also determined. The same sites in L7/L12 were used to attach a rhodamine dye. The formation of ground-state rhodamine 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 free protein and in complexes with L10. The three sites in the CTD, but not the N-terminal sites, cross-linked to L2 and L5 and to 30S proteins S2, S3, S7, S14, and S18 in a manner influenced by elongation factors. Binding to the ribosome and, therefore, function were blocked by zero-length cross-linking within the NTD, but not the CTD. Binding also disrupted rhodamine dimers in the NTD. No rhodamine dimers formed in the CTD.
Traut
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RR
Department of Biological Chemistry, School of Medicine, University of California, Davis 95616, USA.
Dey
D
D
Bochkariov
D E
DE
Oleinikov
A V
AV
Jokhadze
G G
GG
Hamman
B
B
Jameson
D
D
D000577:Amides; D001386:Azides; D001426:Bacterial Proteins / Q000235:genetics*; D003432:Cross-Linking Reagents; D003545:Cysteine / Q000737:chemistry*; D004926:Escherichia coli / Q000235:genetics*; D005456:Fluorescent Dyes; D014644:Genetic Variation; D016297:Mutagenesis, Site-Directed; D017434:Protein Structure, Tertiary; D011725:Pyridines; D012269:Ribosomal Proteins / Q000235:genetics*; D013053:Spectrophotometry; D013439:Sulfhydryl Reagents
D000577:Amides; D001386:Azides; D001426:Bacterial Proteins; D003432:Cross-Linking Reagents; D005456:Fluorescent Dyes; D011725:Pyridines; D012269:Ribosomal Proteins; D013439:Sulfhydryl Reagents; C077391:N-(4-(4-azidobenzamido)butyl)-3-(2'-pyridyldithio)propionamide; D003545:Cysteine