Reaction of N-(3-Pyrene)maleimide with Thiol Groups of Reticulocyte Ribosomes

Eur. J. Biochem. 66, 105-114 (1976)

Reaction of N-( 3-Pyrene)maleimide with Thiol Groups of Reticulocyte Ribosomes Terry LEE and Roger L. HEINTZ

Department of Biochemistry and Biophysics, Iowa State University, Ames, Iowa

(Received October 7, 1975/ March 19, 1976)

The reaction of N-(3-pyrene)maleimide with thiol groups of rabbit reticulocyte ribosomes offers a possible fluorescent probe for studying ribosomal structure and conformation. At relatively low concentrations of N-(3-pyrene)maleimide a group of 30 - 40 readily reactive sulfhydryl residues is derivatized. The major ribosomal proteins containing these thiol groups are identified as S2 + S 3, S5, S7, S8, S29, S31, S32, L1, L5, L6, LlO+L14, L15, L18+L19, and L36. Ribosomal activity, as measured by the nonenzymic binding of phenylalanyl-tRNA and polyphenylalanine synthesis, is inhibited by this degree of reaction with N-(3-pyrene)maleimide. This inhibition is relieved by the prior binding of polyuridylic acid to the ribosomes while the extent of derivatization by N-(3-pyrene)- maleimide is diminished only slightly. The average relative polarization of the fluorescence of the ribosomal bound N-(3-pyrene)maleimide changes significantly with the degree of derivdtization of ribosomal thiol groups or with the binding of polyuridylic acid, indicating the value of such a fluorescent thiol-derivatizing agent as a probe of ribosomal structure.

The ribosome is a very complicated macromolec- ular particle, consisting of at least three RNA species and 55 - 81 different proteins. Because of the central role ribosomes play in protein biosynthesis the structure of these particles and the interactions among the various component RNA and protein species present an intriguing and active area of research [I - 41. The sulfhydryl groups of ribosomal proteins offer a promising target for these investigations for a variety of reasons. These groups are relatively few in number, about 125 per washed reticulocyte ribosome. Of the thiol groups in the intact ribosome only a small percentage react readily with derivatizing agents. For example Acharya and Moore [5] find only 13 of the 39 sulfhydryl groups of native Eschevichiu coli 70 S ribosomes to be available for reaction with 5,5’-dithio-bis(2-nitrobenzoic acid). Un- der appropriate conditions derivatizing agents such as N-ethylmaleimide, 5,5’-dithio-bis(2-nitrobenzoic acid), and iodoacetamide are reasonably specific for thiol residues. The integrity of at least some of these ribosomal sulfhydryl groups appears to be essential for the functioning of ribosomes in protein biosynthesis [6-81. With E. coli ribosomes the thiol groups whose substitution by derivatizing agents leads to inhibition of protein synthesis are located in the

Abbreviurion. MalNEt, N-ethylmaleimide

30-S subunit [7], while in mammalian ribosomes the essential thiol groups lie in the 60-S subunit [9].

Various workers find differences in the reactivities of ribosomal sulfhydryl residues when comparing subunits with the intact monoribosome [5,10,11]. Indeed in several cases the sum of the reactive thiol groups of the larger and smaller subunits is less than that found in the intact monoribosome. This strongly suggests that significant ribosomal conformational changes occur during or subsequent to ribosomal dissociation. This idea is confirmed by experiments which show that different ribosomal proteins are derivatized when monoribosomes react with thiol reagents than when the individual subunits react [lo, 121. Similar results are found in studies involving the lactoperoxidase-catalyzed iodination of E. coli ribosomal proteins [13]. Such probes of ribosomal conformation have been extended to monoribosomes in various stages of peptide bond formation [I 1,121. Rat liver ribosomes carrying peptidyl-tRNA ii7 the acceptor site exhibited less activity toward N-ethylmaleimide (41 out of a total of 120 thiol groups derivatized) than did translocated ribosomes with the peptidyl-tRNA in the donor site (72/120) [ I l l . This result was interpreted as an opening or loosening of the ribosomal structure upon translocation, sub- stantiating earlier studies using sedimentation, hydrogen exchange, and diffusion [15,16]. A finer

106 A Fluorescent Probe of Ribosomal Conformation

delineation of the role of various sulfhydryl groups in ribosomal functioning may be made when the derivatized ribosomal proteins are identified [8,12,17]. Moore [I21 has identified four E. coli ribosomal proteins from the 30-S subunit (S 1, S4, S 18 and S 21) whose derivatization by N-ethylmaleimide is associated with the loss of functional activity. In particular the reaction of N-ethylmaleimide with protein S 18 parallels the loss of activity.

The overall objective of the present research is to obtain quantitative information about the thiol groups of reticulocyte ribosomal proteins which are directly or indirectly involved in protein biosynthesis. The thiol derivatizing agent, N-(3-pyrene)maleimide, whose adduct to sulfhydryl groups is fluorescent, was chosen because of the great potential use of such fluorescent probes in gaining information about the conformational states of macromolecules [18]. For examples of the use of fluorescent probes in studying ribosomal structure see the work of Cantor and his colleagues [2- 41. Also N-(3-pyrene)maleimide itself is non- fluorescent and the sensitivity of detection of the fluorescence of its sulfhydryl group derivative starts to approach that of isotopic methods. From the properties of this fluorescence some information about the microenvironment of the various derivatized ribosomal proteins may be obtained. For example study of the polarization anisotropy of the fluorescence [18] may indicate the degree of flexibility of the derivatized thiol groups of ribosomal proteins under different conditions.

MATERIALS AND METHODS

Preparation und Analysis of Rabbit Reticulocyte Ribosomes and Activity Assays

Deoxycholate-washed reticulocyte ribosomes and crude elongation factors (AS 70 enzyme) were prepared as described by Arlinghaus et al. [19]. Ribo- somal subunits were prepared by a modification of the method of Sherton and Wool [21]. Washed reticulocyte ribosomes (4.5 mg/ml) were incubated in a solution of 50 mM Tris . HCl, pH 7.5, 0.5 M KCI, 3 mM MgC12, 0.5 mM puromycin and 3 mM dithiothreitol for 15 min at 37 "C. The ribosomal subunits were separated on a 5 - 25 % linear sucrose gradient containing 50 mM Tris . HCI, pH 7.5, 0.5 M KCl, 1.5 mM MgC12 and 30 mM mercaptoethanol by centrifugation at 51 5OOxg for 10.5 h in a Spinco SW 25.1 rotor. After centrifugation the bottom of the tube was punctured, l-ml fractions collected and the absorbance at 260 nm determined. The fractions containing the 60-S and 40-S subunits were separately pooled, the concentration of magnesium chloride raised to 20 mM, and the subunits collected by centrifugation at 78500xg for 15 h. The ribosomal

pellets were rinsed and suspended in 0.25 M sucrose at a subunit concentration of 4- 12 mg/ml. These subunit preparations may be stored at -20 "C. Subunits prepared in this manner were essentially free from cross contamination as measured by the require- ment for both 6 0 3 and 40-S subunits for polyphenylalanine synthesis and by their ribosomal RNA content (18-S RNA in the 40-S fraction and 28-S and 5-S RNA in the 60-S preparation).

Ribosomal proteins were prepared essentially as described by Spitnik-Elson [22]. The ribosomal subunit pellet from a high-speed centrifugation (7800 x g , 15 h) was suspended by homogenization in a solution containing 3 M LiCl and 4 M urea at a concentration of 1-4 mg/ml. The resulting suspension was shaken for 48 h at 4 "C and the precipitated RNA removed by centrifugation (I0000 x g , 30 min). The supernatant solution containing ribosomal protein was dialyzed against 8 M urea for at least 6 h at 4 "C and stored at - 20 "C until used. Two-dimensional polyacrylamide gel electrophoresis was performed by the methods of Kaltschmidt and Wittmann [23]. The first-dimension disc gels were run at pH 8.6 in a 6 % polyacrylamide gel at 4.5 mA/gel for 14 h at 4 "C. The sample gel [24] consisted of 0.3 rnl of the ribosomal protein preparation (0.3 mg of 4 0 4 protein or 0.4 mg of 60-S protein in 8 M urea) and 0.1 ml of 2 2) agarose. This sample gel is placed 9.5 cm from one end of the cylindrical gel (18 cm total length). The second dimension separation is performed at pH 4.2 in 18 polyacrylamide gel at 125 V for 24 h at room temperature. The separate ribosomal proteins were located by staining with 0.1 :( Coomassie blue in 7.5 '%, acetic acid and 50 o/, methanol for 4 h. The gels were destained by diffusion in 7.5 x acetic acid and 50'x methanol for 24 h (2 changes of 6 1 each) at room temperature. For determination of radioactivity in the separated ribosomal proteins, the stained spots were cut from the gel slab using a no. 2 cork-borer or a razor blade, placed in a scintillation vial, and 0.3 ml of Protosol and 10 ml Omnifluor (Packard) scintillation 'cocktail' added. A few similar sized pieces of gel were excised at random and used as controls. The vials were tightly capped and incubated at 37 "C for 17 h. After being kept in the refrigerated counter for at least 3 h, radioactivity of the samples was measured using a Packard Tricarb liquid-scintillation spectrometer (model 577). The counting efficiency was approximately 80% and 31 yi for I4C and 3H, respectively.

['4C]Phenylalanyl-tRNA was prepared by the method of Haskinson and Khorana [20] using yeast tRNA and crude yeast aminoacyl-tRNA synthetases. The nonenzymic binding of phenylalanyl-tRNA to the reticulocyte ribosomes was performed as described by Heintz et al. [25]. The reaction mixture (total voluine of 0.3 ml) contained: 67 pM L-phenylalanine, 0.67 mM dithiothreitol, 6.7 mM KCl, 33 mM Tris

T. Lee and R. L. Heintz 107

. HC1, pH 7.5, 13 mM MgClz, 67 pg/ml polyuridylic acid, 70 pg/ml ['4C]phenylalanyl-tRNA (specific activity of 50 mCi/mmol) and 1 mg/ml washed reticulocyte ribosomes. After gentle but complete mixing this solution was incubated for 10 min at 37 "C. The reaction was terminated by chilling (4 "C) and dilution by 10 volumes of cold nonenzymic buffer (33 mM Tris . HCI, pH 7.5, 6.7 mM KCl, and 13 mM MgCL). The ribosomal bound ['4C]phenylalanyl-tRNA was then measured by the cellulose nitrate filtration technique of Nirenberg and Leder [26].

Polyphenylalanine synthesis was measured by a modification of the procedure of Gregg and Heintz [27]. The reaction mixture (total volume of 0.3 ml) contained: 67 pM L-phenylalanine, 1.3 mM GTP, 1.3 mM dithiothreitol, 67 mM KCI, 6.7 mM MgCl2, 33 mM Tris-HC1, pH 7.5, 67 pg/ml polyuridylic acid, 70 pg/ml ['4C]phenylalanyl-tRNA, 0.6 mg/ml AS 70 enzyme fraction, and 40 pg/ml washed reticulocyte ribosomes. After incubation for 1.5 min at 37 'C the reaction was terminated by the addition of 2 ml of cold (4 "C) 5 % trichloroacetic acid containing 1 mM L-phenylalanine. After 30 min in an ice-bath this mixture was heated for 20 min at 90 "C, rechilled and filtered through nitrocellulose membranes. The filtered precipitate was washed 3 times with 3 ml, each, of cold 5 trichloroacetic acid. ['4C]Phenylalanine in both the nonenzymic binding and polyphenylalanine synthesis assays was determined as described pre- viously [25].

Reaction of Ribosomal Tlziol Groups rvitli N-Pyrenemaleimide or N-Ethylmaleimide

The chemical synthesis of N-(3-pyrene)maleimide was accomplished as described by Weltman et ul. [28]. A solution of 8.3 mmol of 3-aminopyrene in 21 ml ice-cold tetrahydrofuran was mixed with 8.8 mmol of maleic anhydride in 10.6 ml ice-cold tetrahydrofuran. After stirring for 17 h at 4 "C the yellow-green precipitate was collected by vacuum filtration. This precipitate [crude N-(3-pyrene)maleamic acid] was washed extensively with tetrahydrofuran and dried. N-(3-Pyrene)maleamic acid (2.1 mmol) was added to a solution of a 40-fold excess of acetic anhydride containing sodium acetate (12.3 mmol). This mixture was stirred at 100 "C for 45 min, cooled to room temperature in a cold water bath, and poured into 45 ml of ice water. The precipitated N-(3-pyrene)maleimide was collected by vacuum filtration, washed first with ice-cold water and then with hexane and dried. After being recrystallized twice by addition of water to an ethanolic solution of the product, the N-(3-pyrene)maleimide was characterized by nuclear magnetic resonance and ultraviolet spectra. A 30 mM stock solution was made in dimethylsufoxide and stored in the dark. The small amount of dimethyl-

sulfoxide carried over into reaction mixtures with reticulocyte ribosomes had little effect on ribosomal activity while other solvents, acetone and dioxane, inhibited the ribosomes significantly (unpublished results). Radioactive N-(3-pyrene)maleimide was synthesized in a similar manner except that [I ,4-14C]- maleic anhydride (specific activity 2.5 pCi/mmol, Amersham/Searle) was used.

The reaction of reticulocyte ribosomes with N-(3- pyrene)maleimide or N-ethylmaleimide (MalNEt) was performed in nonenzymic binding buffer (33 mM Tris-HC1, pH 7.5, 6.7 mM KCl, and 13 mM MgClz). Ribosomes (2 mg/ml) were incubated with N-( 3- pyrene)maleimide or MalNEt (0.2 mM unless indicated otherwise) for 10 min at 37 "C. This time was sufficient for the reaction to reach a plateau. The reaction was terminated by the addition of a 1000-fold excess of 2-mercaptoethanol and the mixture passed over a small column of Sephadex G-25 equilibrated with nonenzymic binding buffer to remove N-(3- pyrene)maleimide, MalNEt, 2-mercaptoethanol, and their adducts. A fresh column of Sephadex G-25 was used each time as it was difficult to wash unreacted N-(3-pyrene)maleimide from the column. Fluorescence intensity was measured using a laboratory-built spectrofluorimeter or a Perkin-Elmer Model MPF 3 L spectrofluorimeter. Polarization measurements of the fluorescence [29] were performed on the latter in- strument using excitation at 342 nm and emission at 376 nm and 395 nm. All spectra were corrected for background fluorescenqe. The measurements were routinely made at 7 C using a 10-mm cuvette. In some experiments the relative polarization values so obtained were compared with those at 37 "C. The 14 C-labeled N-(3-pyrene)maleimide or [3H]MalNEt adducts were measured by hot trichloroacetic acid precipitation as described above for the polyphenylalanine synthesis assay. The reaction of isolated ribosomal subunits with N-(3-pyrene)maleimide or MalNEt was performed as described above using 1.25 mg/ml of the 60-S subunits or 0.75 mg/ml of the 40-S subunits.

RESULTS AND DISCUSSION

Characteristics oj the N-(3-Pyrene)maleimide Derivatization of Reticulocyte Ribosomes

The reaction of reticulocyte ribosomal thiol groups with N-(3-pyrene)maleimide as a function of the N-(3-pyrene)maleimide concentration is illustrated in Fig. 1. The rapid rise in fluorescence intensity at low concentrations of N-(3-pyrene)maleimide followed by a more gradual increase at higher concentrations of the derivatizing agent suggests that a relatively sensitive class of sulfhydryl groups are derivatized at quite low concentrations of N-(3-pyrene)maleimide


n I I I I 0 "0 0.1 0.2 0.3 0.4 0.5

N - (3-Pyrene)maleimide (mM)

Fig. 1, Derivatization of' ribosomal tliiol groups at various concentrations of N-(3-pyrene)maleimide. Washed reticulocyte ribosomes (2 mg/ml) are reacted with the indicated concentrations of N-(3- pyrene)maleimide in the nonenzymic binding buffer (33 mM Tris . HCI, pH 7.5,6.7 mM KC1 and 13 mM MgCll) for 10 min at 37 'C. The reaction is stopped by addition of 1000-fold excess 2-mercaptoethanol and the reaction mixture (0.6 ml) passed over a column of Sephadex G-25 (Materials and Methods). The relative intensity (0) of the emitted fluorescence is measured at 395 nm with excitation at 342 nm. Activity of the ribosomes is measured directly on aliquots of the N-(3-pyrene)maleiniide reaction mixture as described in Materials and Methods. These activities, nonenzymic binding of [14C]phenylalanyl-tRNA (0) and polyphenylalanine synthesis (A) are expressed as a percentage of the activity lost compared to control ribosomes [minus N-(3-pyrene)maleimide]. Typical values of the assays would be 400 pmol of [14C]phenylalanine (from ['4C]phenyIalanyl-tRNA) incorporated into polyphenylalanine/mg ribosomes

followed by a class of thiol groups reactive at only relatively high N-(3-pyrene)maleimide concentrations (> M). Derivatization of the more highly reactive class of thiol groups leads to extensive inhibition of ribosome1 activity as measured by the polyuridylic acid directed nonenzymic binding of phenylalanyl- tRNA or polyphenylalanine synthesis. This inhibition of ribosomal activities is not due to a shift of the magnesium ion optimum for the N-(3-pyrene)maleimide-substituted ribosomes, since partially inhibited ribosomes display the same Mg2+ optimum, albeit with lower activity, as do control ribosomes. Neither is this inhibition due to the presence of the small amount of dimethylsulfoxide included from the stock solution of N-(3-pyrene)maleimide as similar amounts of dimethylsulfoxide alone have no effect on these ribosomal activities. Derivatization of reticulocyte ribosomal sulfhydryl groups with N-(3-pyrene)maleimide does not lead to dissociation of the 80-S monoribosomes into subunits nor does treatment with this reagent prevent such a dissociation (unpublished results).

As it is difficult to quantitatively equate the number of thiol groups derivatized with the fluorescence intensity of the N-(3-pyrene)maleimide adduct, 14C- labeled N-(3-pyrene)maleimide was synthesized and

-0 0.5 1 .o 1.5 2 .0 N- (3-Pyrene)maleimide (mM)

Fig. 2. Estimation oJthe number of't.ihosomal tliiol groups derivarized at various N-(3-pyrene)rnaleimide concentrations. '4C-labeled N-(3- pyrene)maleimide (specific activity 11 1 pCi/mmol) is synthesized by the methods of Weltman et ul. [26]. This compound (Materials and Methods) is used to derivatize washed reticulocyte ribosomes as described in Fig. 1. Aliquots of the ribosome peak (void volume) of the Sephadex (3-25 column are taken to measure the 14C-labeled N-(3-pyrene)maleimide bound. This is accomplished by the hot trichloroacetic acid precipitation of ribosomal proteins as described for t.he polyphenylalanine synthesis assay (Materials and Methods). The results are expressed as mol I4C-labeled N-(3-pyrene)maleimide bound/mol ribosomes, calculated using the specific activity of the radioactive N-(3-pyrene)maleimide, the counting efficiency (80 %) of the liquid-scintillation spectrometer, the absorbance at 260 nm of the ribosome peak (11.2 absorbance units/mg), and a molecular weight of 4.0 x lo6 for the ribosomes

used in a similar experiment (Fig. 2). The more readily reactive class of ribosomal thiol groups appears to number about 30/ribosome (mol/mol). The continued derivatization at higher N-(3-pyrene)maleimide concentrations results in the addition of over 100mol N-(3-pyrene)maleimide/mol ribosomes (2 mM N-(3- pyrene)maleimide). Experiments in our laboratory using 5,5'-dithio-bis(2-nitrobenzoic acid) show that these washed reticulocyte ribosomes contain approximately 125 reactive sulfhydryl groups (results not shown). These experiments are carried out in 8 M urea to denature the ribosomes and so render all of the sulfhydryl groups available for reaction with the derivatizing agent. With intact ribosomes only 15 thiol groups per ribosome readily react with 5,5'-dithio- bis(2-nitrobenzoic acid). This is surprising in view of the results obtained with N-(3-pyrene)maleimide (Fig. 1 and 2) where more than twice this number of sulfhydryl groups react at low concentrations of N-(3-pyrene)maleimide. Such results illustrate the difficulty of interpreting the 'availability' of ribosomal thiol groups to mean such groups are on or near the surface of the ribosome or at least available to the solvent [30]. Each thiol residue in the complicated structure of the ribosome has a microenvironment unique to itself. Therefore each sulfhydryl group


I

0.5 1 .o 1.5 2 .o [MalNEt] (mM)

Fig. 3. Derivatization of ribosomal t/?io/ groups at various concentrations of 3H-laheled N-ethylmaleimide. Reticulocyte ribosomes (2 mg/ml) are reacted with the indicated concentrations of [3H]MalNEt (specific activity, 230 mCi/mmol, New England Nuclear) in a buffer containing 33 mM Tris . HCI, 6.7 mM KCI and 13 m M MgCL for 10 min at 37 "C. The amount of [3H]MalNEt bound to the ribosomes (0) is determined as described in Fig. 2 for ''C-labeled N-(3-pyrene)maleimide. The counting efficiency of the [3H]MalNEt was 31 %. Aliquots of the MalNEt reaction mixture are used directly (Fig. I ) to measure ['4C]phenylalanyl-tRNA binding (0) and [14C]polyphenylalanine synthesis (A). For these samples containing both 3H and 14C, 3H is counted (Packard Tricarb Model 577 Liquid-Scintillation Spectrometer) at 35 %gain in a window of 50-300 and corrected for 14C radioactivity. I4C is measured at 1 8 % gain in a window of 400-1000 at which settings "1 radioactivity was negligible. The results are expressed as a percentage of control ribosomes (minus MalNEt) as in Fig. 1

may have a different reactivity towards derivatizing agents in general and may behave differently towards different reagents than the other sulfhydryl groups of the ribosome. However, changes in the reactivity of specific thiol groups toward a given reagent under identical derivatizing conditions would indicate a conformational change of the ribosome as reflected by a change in the microenvironment of that particular thiol group. The number of thiol groups which are readily reactive with low N-(3-pyrene)maleimide concentrations is in reasonable agreement with the number of 'available' sulfhydryl groups on rat liver ribosomes [l I]. The observation that derivatization continues with increasing concentrations of N-(3-pyrene)maleimide until a large proportion of the total sulfhydryl groups of the reticulocyte ribosomes has reacted suggests that the bulky, hydrophobic pyrene side- group of N-(3-pyrene)maleimide may be disrupting the conformation of the ribosomes and their con- stituent proteins, thus making 'available' for reaction thiol groups which were 'buried' or unreactive because of their microenvironment. In agreement with this idea the use of a similar derivatizing agent, MalNEt, which has a less bulky hydrophobic side-chain, results in the reaction of approximately 30 sulfhydryl groups at low MalNEt concentrations (Fig.3). In contrast to N-(3-pyrene)maleimide higher concentrations of MalNEt do not lead to further derivatization but the number of thiol groups reacted reaches a plateau. The number of ribosomal thiol groups reacting at the lower concentrations of reagent are quite similar for N-(3-pyrene)maleimide and MalNEt and the loss of activity with derivatization of these

readily reactive sulfhydryl groups is essentially identical for these two compounds (compare Fig. 2 and 3). A further indication that N-(3-pyrene)maleimide and MalNEt at low concentrations are reacting with the same ribosomal thiol residues lies in the observation that prior treatment of ribosomes with N-(3-pyrene)- maleimide or MalNEt allows little or no subsequent reaction with the other compound (results not shown).

Identification of the Readily Reactivr Ribvsomal Thiol Groups

[3H]MalNEt is used in place of N-(J-pyrene)- maleimide in experiments to identify the derivatized ribosomal proteins because while 14C-labeled N-(3- pyrene)maleimide was synthesized and used in binding experiments (Fig.2), its specific activity was too low to measure accurately its attachment to individual ribosomal proteins. Reticulocyte ribosomes are reacted with [3H]MalNEt and then dissociated into 60-S and 40-S subunits by appropriate adjustment of the KC1 and MgCL concentrations. These subunits are separated via sucrose gradient centrifugation. Ribo- somal proteins are then prepared from the separated subunits and analyzed by two-dimensional polyacrylamide gel electrophoresis as described in the Materials and Methods section. On the 40-S ribosomal subunit seven or eight proteins, S2 + S 3 , S5, 57, SS, S29, S31 and S32, contain most of the N-(3- pyrene)maleimide-derivatized thiol groups (Fig. 4). S2 + S3 and S5 are especially highly labeled, ac- counting for about half of the protein-bound MalNEt. The proteins of the 60-S subunit most reactive with

110

36

32

28

24

- e m 8 h * > ._ ._ c

5 16 d

12

e

4

C I 1 2+3 4

A Fluorescent Probe of Ribosomal Conformation

6 8 10 12 14 16 18 x) 22 24 26 28 30 32 40-S Subunit proteins

Fig. 4. Identification of the proteins of the 40-S ribosomal subunits which are readily reactive w'ith 3H-labeled N-ethylmaleimide. Reticulocyte ribosomes (2 mg/ml) are incubated with 0.2 mM t3H]MalNEt as described in Fig. 3. The reaction mixture is layered on a dissociative sucrose gradient (5-25 sucrose, 50 mM Tris . HC1, pH 7.5,0.5 M KCI, 1.5 mM MgClz and 30 mM 2-mercaptoethanol) and centrifuged 51 500 x g for 10.5 h in a Spinco SW25.1 rotor (Materials and Methods). The separated 60-S and 40-S subunits are identified by the pattern of absorbance at 260 nm. Fractions containing the large and small subunits respectively are pooled, the MgCb concentration is raised to 20 mM, and the subunits collected by centrifugation at 78 500 x g for 15 h. t3H]MalNEt labeled proteins of the 4 0 3 subunits are prepared and analyzed by two- dimensional polyacrylamide gel electrophoresis as described in the Materials and Methods section. The results are expressed as a percentage of the total [3H]MalNEt bound to the 4 0 3 subunit proteins found in each of the individual 40-S protein spots

MalNEt under these conditions are L1, L5, L6, L10 + L14, L18 + L19 and L36. L1 alone contains about 26 % of the labeled MalNEt (Fig. 5).

Reticulocyte ribosomal subunits prepared by our procedure (Materials and Methods) are essentially free of cross contamination by two criteria. Neither the 60-S nor the 40-S subunits from control ribosomes are active in polyphenylalanine synthesis by themselves but are highly active when combined. Also the isolated 40-S subunits contain only 18-S RNA while the 60-S subunit preparation exhibits only 28-S and 5-S RNA as judged by sucrose density gradient centrifugation. The pattern of proteins on the two-dimensional gels is numbered, using the prefixes S and L for the 40-S and 60-S subunits, respectively, according to the convention of Wittmann et al. [31]. The array of proteins on the polyacrylamide slab for both the 40-S and 60-S subunits appears very similar to that observed by Sherton and Wool [21] for rat liver ribosomal subunits. The number of protein spots (33 and

48 for the 40-S and 6 0 3 subunits respectively) is also similar to those found by other workers using rabbit reticulocyte ribosomal subunits prepared in slightly different manners [32 - 341. Complete quantitation of the results of labeling the ribosomal proteins with rH]MalNEt is difficult for several reasons. Some labeled protein is lost during subunit preparation as has been observed by other workers [12,35]. Some ribosomal protein appears to aggregate and not migrate from the origin during the two-dimensional gel electrophoresis. Counting conditions, quenching and spurious counts are not exactly uniform and are difficult to correct. Nevertheless if one assumes that the label contained by L5 and S32 constitutes a unit quantity, one derivatized thiol group per protein, and uses only the significantly labeled proteins mentioned above, one may cal- culate that 40-S and 60-S subunits contain approximately 17 and 15 derivatized thiol groups, respectively. The sum of these (32) is in good agreement with the

T. Lee and R. L. Heintz

28

111

Fig. 5. Identijkation of the proteins of'the 60-S ribosomal suhunils which are readily reactive with 3H-iaheted N-ethyimateimide. Reticulocyte ribosomes are reacted with [3H]MalNEt, 60-S subunits prepared and the 60-S ribosomal proteins analyzed as described in Fig. 5. The results are expressed as a percentage of the total [3H]MalNEt bound by the 60-S ribosomal subunit proteins found in each of the individual 60-S protein spots

amount of label expected on the intact 80-S ribosomes by thiol derivatizing agents, N-(3-pyrene)maleimide or MalNEt (Fig. 3).

Inhibition of the ability of reticulocyte ribosomes to bind phenylalanyl-tRNA or synthesize polyphenylalanine is the result of substitution of thiol groups in the 60-S subunit (results not shown [9]). The concentration dependence [N-(3-pyrene)maleimide or MalNEt] of the inactivation of the 604 subunit parallels that of the intact 80-S ribosomes. The same results are obtained whether the 80-S ribosome is reacted with N-(3-pyrene)maleimide or MalNEt and then the subunits prepared or whether the isolated subunits are treated directly with the derivatizing agent (results not shown). Inclusion of phenylalanyl- tRNA and poly(U) in the reaction mixture with N-(3-pyrene)maleimide affords almost complete protection against the loss of activity (Fig. 6). Analogous results are obtained in experiments in which N-(3- pyrene)maleimide is replaced with MalNEt. Similar results have been observed by other workers [8]. However, in contrast to their results poly(U) alone accounts for most of this protective effect. Cheng and McAllister report a decrease in the reactivity of MalNEt [in the presence of poly(U) and phenylalanyl-tRNA] with one particular protein band from the 60-S subunit on disc gel electrophoresis. In our experiments when ribosomes are reacted with [3H]- MalNEt in the presence or absence of poly(U) plus phenylalanyl-tRNA or with poly(U) alone, we observe

100

80

- x 60 - - >

u - - c .- v) 4c 0 i

x

C

Reaction time (rnin)

Fig. 6. Protection of' ribosomes against N-(3-p~~rcne)mai~imide inactivation by polyjuridylic acid). Washed reticulocyte ribosomes (2 mg/ml) are incubated alone (A), with poly(U) (140 kg/ml) (0) or with poly(U) (140 kg/ml) and phenylalanyl-tRNA (400 pg/ml) (0) in the nonenzymic binding buffer (Fig. 1) for 8 min at 37 "C. N-(3-Pyrene)maleimide (0.2 mM) is added and the incubation continued for the indicated times at 37 "C. Ribosomal activity is measured by the polyphenylalanine synthesis ascay (Fig. 1). The amount of ['4C]phenylalanyl-tRNA bound to the ribosomes under the preincubation conditions routinely is about 40 pmol/mg ribosomes. The results are expressed as a percentage ofcontrol ribosomes incubated in a similar manner except that no N-(3-pyrene)maleimide was added. Such an incubation has little or no effect on the activity of the ribosomes. Thecontrol ribosomes have an activity of 500 pmol ['4C]phenylalanine as polyphenylalanine/mg ribosomes


Table 1. The relative devivatizafion of individual ribosomal proteins in the presence of poly( U ) andphenylalanyl-tRNA Washed reticulocyte ribosomes (2 mg/ml) are preincubated for 8 min at 37 "C in the nonenzymic binding buffer alone (column 2), with 200 pg/ml poly(U) (column 3) or with 200 pg/ml poly(U) and 67 pg/ml phenylalanyl-tRNA. [3H]MalNEt (0.045 mM) is added and incubation continued for 10 min at 37 "C. Ribosomal proteins were analyzed as described for Fig.4 and 5. The results are expressed as a percentage of the total [3H]MalNEt bound to the 4 0 3 subunit (designated S) proteins and the 60-S subunit (designated L) proteins. Only those ribosomal proteins containing significant amounts of [3H]MalNEt (greater than 2j/, of the total for each subunit) are included for comparison

Ribosomal Incorprorated N-ethylmaleimide arotein

ribosomes ribosomes ribosomes

+ phenylalanyl- tRNA

+ POlY(U) + POlY(U)

of total

s 2 + 3 s 5 s 7 5 8 S18 S 29 S 31 S 32 s 33 L1 L5 L6 L 7 L10 + 14 L15 L18 + 19 L 36

15.5 34.8 3.2 3.6 0.1 5.5 4.3 5.8 2.0

26.3 4.3 3.4 2.3 8.2 3.7 9.0 1.2

9.0 36.1 4.6 3.2 4.6 9.7 1.1 5.1 6.8

26.2 4.0 5.8 1.6 8.8 5.6 6.8 8.2

11.2 41.1

5.0 3.8 0.4 9.3 1.9 3.0 0.8

30.1 6.2 0 4.5 9.8 3.2

10.0 7.6

little or no change of the amount of label in any of the 60-S subunit proteins (Table 1). Surprisingly, three proteins, S2+S3,S31 and S32 of the 40-S subunit do exhibit some decreased reactivity (x) with [3H]- MalNEt in the presence of poly(U) plus phenylalanyl- tRNA. Interpretation of such results is difficult for two major reasons. Absolute quantitation of the recoveries of radioactive proteins on the two-dimensional gels is difficult. Also only about 15% of the ribosomes in these preparations exhibit the ability to bind phenylalanyl-tRNA. More ribosomes may bind poly(U) alone but this has not been estimated owing to low but significant nuclease activity. There- fore the methods used may not be sensitive enough to observe the small differences expected.

The Polarization of the Fluorescence of Ribosomal-Bound N- (3-Pyrene) maleimide

The polarization of the fluorescence of ribosomal- bound N-(3-pyrene)maleimide may provide some

Table 2. The relative polarization of the jliiorescence of ribosomal bound N- (3-pyrene) maleimide Washed reticulocyte ribosomes (2 mg/ml) are incubated for 10 min at 37 "C with the indicated concentrations of N-(3-pyrene)nialeimide in the nonenzymic binding buffer (Fig. 1 ; Materials and Methods). Three separation incubation mixtures contain: ribosomes alone (columns 2 and 3), ribosomes plus poly(U) (67 pg/ml) (columns 4 and 5)> or ribosomes plus poly(U) (67 pg/ml) and phenylalanyl- tRNA (200 pg/ml) (columns 6 and 7). The fluorescence intensity and relative polarization of the emitted fluorescence are measured as described in Fig. 1 and the Materials and Methods section. I represents the relative fluorescence intensity of ribosomal bound N-(3-pyrene)maleimide. P is the relative polarization of the fluorescence of ribosomal-bound N-(3-pyrene)maleimide

Ribosomes N-(3- Ribosomes Ribosomes

malei- + phenyl- mide aianyl-tRNA

Pyrene)- + POlY(U) + POlY(U)

I P I P I P

mhl

0.01 110 0.27 90 0.21 50 0.21 140 0.20 115 0.21 0.02 130 0.28

0.035 200 0.24 130 0.20 170 0.23 0.05 410 0.24 310 0.19 300 0.22 0.10 500 0.21 430 0.19 380 0.22 0.15 580 0.21 500 0.21 400 0.20 0.51 640 0.20 530 0.15 440 0.20

information on the microenvironment of the derivatized sulfhydryl groups. Changes in the relative polarization of such fluorescence can offer a crude measure of ribosomal conformational changes. As the extent of derivatization of ribosomal thiol groups is increased with increasing concentrations of N-(3- pyrene)maleimide the relative polarization of the fluorescence drops (Table 2). This decrease in the polarization value indicates an average less restricted microenvironment for the derivatized sulfhydryl groups. This observation may merely be a reflection of the location of the less reactive thiol residues in less rigid portions of the ribosomal structure. A much more likely explanation is that an opening or relaxation of the ribosomal structure occurs with increasing reaction of ribosomal thiol groups with N-(3-pyrene)- maleimide. This interpretation is consistent with the continued substitution of ribosomal thiol residues at high concentrations of N-(3-pyrene)maleimide as opposed to the reaction with MalNEt (Fig. 1 - 3). Additional supportive evidence comes from the observation that the relative polarization of the fluorescence of N-(3-pyrene)maleimide-derivatized sulfhydryl residues of the reticulocyte ribosomes is more affected by temperature differences (7 "C vs 37 "C) at high N-(3-pyrene)maleimide concentrations than at low concentrations. Also the relative polarization values of N-(3-pyrene)maleimide-derivatized ribosomal subunits decrease with increasing KC1 concen-


trations at a variety of MgC12 concentrations (results not shown). Such an increase in the K + : Mg2+ ratio would favor a loosening or expansion of the ribosome structure [36]. The 40-S subunit is especially sensitive in this regard and so exhibits a larger decrease in the relative polarization of the fluorescence of its N-(3- pyrene)maleimide-labeled thiol groups at high K' : Mg2' ratios than does the 60-S subunits. These results emphasize the importance of hydrophobic interactions in the structure of ribosomes. Although obvious from the dissociative effect of detergents on ribosomal proteins and RNA, the contribution of hydrophobic associations is often overlooked. Probes such as N-(3-pyrene)maleimide may prove useful in investigating such interactions.

The interpretation of the polarization data (Table2) as an increase in the rotational freedom of the ribosome-bound fluorophore is only tentative. Similar results might be obtained by the occurrence of energy transfer between adjacent dye molecules or an increase in the excited state lifetime of the fluorophore. Further experimentation such as quantitation of polarization at low temperatures or as a function of temperature and viscosity should discern among these possibilities.

Table 2 also shows that the presence of poly(U) in the reaction mixture leads to a lessening of the relative polarization of the fluorescence of ribosomal bound N-(3-pyrene)maleimide even at low concentrations of the reagent. This effect does not seem to be associated with the extent of derivatization of the ribosomal thiols as the fluorescence intensity is decreased only slightly by the inclusion of poly(U) in the reaction mixture. An immediate explanation of these results is that the binding of this messenger RNA causes a relaxing or loosening of the ribosomal structure. This observation would appear to conflict with the results of Vournakis and Rich [16], who interpreted their results to show a lessening in the diameter of the ribosome upon binding messenger RNA. The two results may not be incompatible as their technique measures a gross overall property of the ribosome and the polarization experiment (Table 2) reflects the microenvironment of a relatively small number of thiol groups of the ribosome, certainly only a small portion of the total ribosomal structure. An alternative explanation of these results may be that the decrease in the polarization value reflects an increase in the rotational velocity of the ribosomal particle as a whole. In this case a more compact ribosomal structure as a result of poly(U) binding would exhibit a lessening of the relative polarization of the fluorescence of the N-(3-pyrene)maleimide- derivatized ribosomes. The results of Table 2 qualita- tively illustrate that some conformational change does occur during one stage of the function of reticulocyte ribosomes, the binding of messenger RNA. Fluores-

cent probes such as N-(3-pyrene)maleimide should prove useful in defining structural changes in the ribosomes during other stages of protein biosynthesis. Other studies (labeling of 'available' sulfhydryl residues, sedimentation and hydrogen exchange) indicate that the ribosome structure expands upon translocation [ l l , 15,161. With the use of fluorescent probes more elaborate quantitative data, such as a study of the rate of relaxation of the polarization anisotropy of the fluorescence or energy transfer between two suitable fluorescent molecules [18] need to be collected to describe such ribosomal conformational changes adequately. The utilization of single characterized fluorescent derivatives of particular ribosomal proteins in such experiments would be especially valuable when methods for reconstituting mammalian ribosomes are developed.

The exccllent technical assistance of Ms Marilyn Darkes and the constructive criticism of this manuscript by Dr Jack Horowitz is highly appreciated. This work was supported by a Research Grant (HE 12549) from the Heart and Lung Institute of the National Institutes of Health.

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* To whom correspondence should be addressed

Reaction of N-(3-Pyrene)maleimide with Thiol Groups of Reticulocyte Ribosomes

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