Using the lactoperoxidase-catalyzed iodination system as a probe for exposed proteins, the location of ribosomal proteins on 50-S subunits of Escherichia coli was examined. These studies demonstrated that a t least three of the proteins in the 50-S ribosome are iodinated and may be termed exposed proteins. Most of the proteins, on the other hand, are not iodinated and can be classified as buried or partially buried proteins. Unfolding of the 50-5 ribosome by the removal of Mg2+ resulted in a decreased organization of the ribosome structure and allowed all the proteins except one to be iodinated.The Escherichia coli 50-5 subunit is composed of approximately 34 distinct proteins [l]. The studies of Traut et al. [I] have suggested that, except for two of these proteins, the remaining exist in one copy per ribosome. A question which remains unresolved is the arrangement of these proteins within the subunit structure. Specifically, in the present study the topography of the 50-S subunit has been investigated to establish which proteins are buried and which are exposed or on the surface of the ribosome. To answer this question, advantage has been taken of the lactoperoxidase-catalyzed halogenation system which, under the conditions employed, is a macromolecular probe for exposed proteins in organized macromolecular systems [Z]. MATERIALS AND METHODSThe methionine and arginine-required mutant of E. coli strain K711 (RCrel) was used throughout these studies. The bacteria were grown at 37 "C with shaking in minimal media [3] supplemented with I1 mM glycerol 0.33 mM L-methionine and 0.24mM L-arginine. Bacteria were grown to an absorbance a t 575 nm of 0.1 and incubated for three generations in the presence of tritiated tyrosine to label all proteins uniformly with tritium. The 30-S and 50-S ribosomal subunits were isolated as previously described [4]. The cells, collected by centrifugation, were washed with 10 mM Tris-HC1,6 mM 2-mercaptoethanol, 30nM ammonium chloride buffer pH 7.4, supplemented with 10 nM magnesium acetate and resuspended in 3 ml of Tris-mercaptoethanol-NH4ClEnzymes. Lactoperoxidase (EC 1.11.1.-).32 Eur. J. Uiochem., Vo1.33 buffer plus 1OmM Mg2+. Cells were broken in a French pressure cell a t 6000 to 8000 lb/in2 and the debris removed as previously described [4]. The ribosomes were pursed as outlined earlier [4]. To fractionate monosomes into subunits, approximately 125 Azao units (one A,,, unit is equal t o an absorbance of I a t 260 nm in a cuvette of light path 1 cm) were layered on 27 ml of a linear 5 to ZOO/, sucrose gradient in Tris-mercaptoethanol-NH4Cl buffer plus 10 mM Mg2+ and centrifuged a t 21500 rev./& for 10 h in an SW 25.1 rotor. Gradients were collected through an ISCO ultraviolet Monitor (Instruments Specialties Company) and the peaks corresponding to 30-S and 50-S ribosomal subunits collected. The 50-S fraction was placed in a length of dialysis tubing and concentrated by packing the tubing in fine granular sucrose. The concentrated sample was dialyzed against 50 mM Tris-acetate, 10 m...

Lactoperoxidase-catalyzed iodination of 30-S and 50-S subunits of Escherichia coli ribosomes maintained at 0 "C revealed that proteins L2, L5, LIO, L11, L15, L26, L27, L29 and L30 of the large subunit and S2, S3, S7, S9, S10, S11, S12, S14, S19 and S21 of the small subunit were the most susceptible to iodination. Unfolding of the 50-S subunit by the removal of Mg2+ greatly increased iodination of the majority of the large subunit proteins while comparatively few of the 30-S proteins became more susceptible to iodination under identical conditions. Less extensive conformational changes were observed when the iodination reaction was allowed to proceed at higher temperatures. At 37 "C, significant increases in the iodination of proteins S6, S13, S15, S16, L3, L8, L9, L18 and L19 were noted suggesting that a loosening of the ribosome structure occurs at this temperature. Enzymatic iodination had little effect on the activity of the subunits in vitro with respect to G-factor-dependent GTPase and poly(U)-directed poly(phenyla1anine)-synthesizing activities. The lactoperoxidasecatalyzed iodination system is evaluated as a probe for detecting conformational changes and for determining the topographical nature of the ribosome.Lactoperoxidase-catalyzed iodination of ribosomal proteins was first introduced as a probe for the study of ribosome topography when we reported on the localization of ribosomal proteins in the 50-S subunit of Escherichia coli [l]. The specificity of lactoperoxidase for tyrosine residues and its large molecular weight (78000) makes the enzyme an ideal surface probe since its size should prevent it from penetrating the structure of the ribosome. Preliminary results involving separation of the iodinated proteins by onedimensional polyacrylamide gel electrophoresis revealed that relatively few of the 50-S proteins were susceptible to extensive iodination. These data were confirmed by Litman and Cantor [2] using twodimensional separation of iodinated 50-S proteins. A study by Miller and Sypherd[3] showed the 30-S ribosomal proteins to be iodinated to a greater extent than those of the large subunit.As information on ribosome structure has accumulated, it has become increasingly apparent that the ribosome does not exist as a rigid structure. Ionic conditions and temperature have been shown to influence the conformationalstate of the ribosome [4-71.Probes originally designed to study the location of ribosomal proteins have also been employed to detect conformational changes that ribosomes may undergo. Ginzburg et al. [S] used N-ethylmaleimide to study functional sites and conformational changes occurring under different ionic conditions in 30-S subunits. Conformational changes induced by high concentrations of K + and Na+, as well as by increased temperatures were also detected by differences observed in the reactivity of sulfhydryl groups of ribosomal proteins with 5,5'-dithiobis(2-nitrobenzoic acid) [9]. These studies are limited, however, by the number of sulfhydryl-containing proteins.Although resol...

Using either soluble or solid-state lactoperoxidase, a comparison was made between the enzymic iodination of ribosomal proteins iodinated as 3 0 4 and 50-S subunits or as 7 0 4 monosomes. Proteins S7, S11 and S12 of the 30-S subunit and proteins L2, L11, L26 and L28 of the 50-S subunit were labelled to a greater extent in isolated particles than in the 70-S ribosome. In contrast, proteins S4, S6, S19 and S20 were labelled to a lesser extent in the isolated subunit. No significant differences were observed in the iodination patterns of ribosomes iodinated in the presence of soluble lactoperoxidase and those iodinated in the presence of lactoperoxidase bound to Sepharose 4B. It is suggested that the 30-S subunit undergoes a conformational change during its association with the 50-S subunit to form a 70-S monosome. Implications from results obtained with solid-state lactoperoxidase-catalyzed iodination of ribosomal proteins are also discussed.Enzymic iodination of ribosomal proteins has been employed as a probe for the identification of surface proteins and detection of conformational changes in ribosome structure [l -41. Although a greater majority of 30-S proteins are extensively modified as compared to those of the 5 0 4 subunit, these studies reveal that essentially all of the 30-S and 50-S proteins become labelled, to some extent, with iodide. To eliminate the possibility that the accessibility of the individual ribosomal proteins to iodination is related to the ability of the lactoperoxidase to penetrate the structure of the ribosome, use was made of lactoperoxidase immobilized by covalent coupling to Sepharose 4B. Employing both soluble and immobilized lactoperoxidase, we have compared the ability of these two preparations to iodinate ribosomal proteins of isolated 3 0 3 and 5 0 4 subunits as well as subunit proteins derived from iodinated 70-S monosomes. Differences in iodination patterns between ribosomal proteins derived from isolated subunits and from 70-S ribosomes are also discussed with respect to structural alterations. MATERIALS AND METHODSBacterial strains, ribosome preparations, soluble lactoperoxidase-catalyzed iodinations and extraction of ribosomal proteins were as previously described [I, 51. 70-S monosomes were maintained at 10 mM Mg + throughout these experiments. Preparation of Solid-state LactoperoxidaseSolid-state lactoperoxidase was prepared according to the method described by David [6]. Sepharose 4B (5.0ml packed volume) was mixed with an equal volume of deionized water and activated with 2.5 g of cyanogen bromide; the reaction mixture, maintained at 20 "C, was immediately brought to pH 11 .O by the addition of 2 M NaOH. This pH was maintained by the further addition of 2 M NaOH until completion of the reaction as indicated by stabilization of pH. The temperature then was lowered to 4°C and the reaction mixture transferred to a Buchner funnel and washed under suction with phosphatebuffered saline. Washed Sepharose beads were then combined with purified lactoperoxidase [8] at a concentr...

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