Singlet energy transfer studies of the arrangement of proteins in the 30 S Escherichia coli ribosome

J. Mol. Biol. (1975) 97, 443-470

Singlet Energy Transfer Studies of the Arrangement of Proteins in the 30 S Escherichia coli Ribosome KUEI-HUANG HUANO, ROBERT 1=[. ~AIRCLOUGHAND CHARLESR. CANTOR

Departments of Chemistry and Biological Sciences Columbia University New York, N.Y. 10027, U.S.A. (Received 29 January 1975, and in revised form 5 June 1975) Reassembly in vitro has been used to prepare a set of twenty Escherichia coli 30 S ribosomes containing pairs of proteins carrying different covalently attached fluorescent labels. These 30 S particles have normal sedimentation properties and moderate to excellent activity in polyphenylalanine synthesis. Reassembly of the dye-conjugated proteins proceeds well but exhibits a preference for incorporation of species containing fewer dyes than the average of the population. Singlet-singlet energy transfer measurements have been carried out on all samples. The majority of the results are highly reproducible. A wide range of efflciencies is seen for protein pairs and the results sort roughly into four proximity classes. Assuming a Poisson distribution of dye stoichiometries and either random surface labeling on spherical proteins or specific unique labeling sites, observed effieiencies were analyzed to yield distance estimates for each protein pair. These were also generally quite reproducible for different samples of a given protein pair. The distance estimates show that four protein pairs are so close that extensive, protein-protein contact is likely. Six others are close enough so that these proteins could well be nearest neighbors in the 30 S particle although there is probably intervening RNA. The remainder are far enough apart that other proteins or extensive regions of the 16 S rRNA are likely to lie in between. There is sufficient distance information on six proteins to conclude that their threedimensional arrangement has a marked deviation from planarity. Variations in transfer efficiency as a function of the degree of labeling indicate that $20 protein has a length at least twice what would be expected for the equivalent spherical protein. All of the distance estimates available are highly consistent with other known proximity information. The detailed results suggest that proteins linked by reassembly in vitro are usually close but are not necessarily in direct contact. This would mean that many of the assembly steps are mediated by the 16 S rRNA.

1. I n t r o d u c t i o n A number of different approaches are yielding information on the topological arrangement of proteins in the 30 S Escherichia cell ribosome. I n almost all these approaches what at this stage appears to be a rational simplification of considering each protein as a fundamental unit has been accepted. Thus what is sought is data on relative protein proximity without special concern for the shape or structure of each protein. Crosslinking by bffunctional reagents is a natural approach and has been the one most widely used so far (Bickle et al., 1972; Chang & Flaks, 1972; Lutter et al., 1972; Shih & Craven, 1973; Sun e~ al., 1974). Limited nuclease digestion of 30 S particles 443

444

K . . H . HUANG, R . H . F A I R C L O U G H AND C. R. CANTOR

(Morgan & Brimacombe, 1973) and a variety of chemical modification studies have also yielded useful information. Electron microscopy using immunological markers (Wahl, 1974; Lake et al., 1974; Tischendorf e t a / . , 1974) and neutron scattering of selectively deuterated ribosomes (Moore et al., 1974) both appear v e r y promising although further development is required. However, each of these techniques has potentially serious limitations in addition to the considerable effort in executing a n y of them. I t seems unlfl~ely t h a t a n y single technique is capable of providing a complete, correct topological picture of the arrangement of ribosomal proteins. I n this p a p e r the feasibility of attacking this problem b y singlet-singlet energy transfer between pairs of fluorescently labeled ribosomal proteins will be demonstrated. While this technique also has serious limitations, it offers some unique advantages including the ability to make rapid measurements on dilute solutions of particles which still maintain biological activity. I n an ideal case, energy transfer can provide a specific distance measurement between fixed points in a complex structure (Wu & Stryer, 1972; Beardsley & Cantor, 1970; Bunting & Cathou, 1973). The major hurdles to using this technique on ribosomes are the introduction of labels at specific points and certain technical problems inherent in a system of this size and complexity. These problems have now been largely overcome. E n e r g y transfer has provided estimates of the relative proximity of m a n y pairs of ribosomal proteins. The results obtained are consistent with virtually all of the implications about 30 S structure currently available from other techniques.

2. Materials and Methods (a) Mater~,~ FITCT was purchased from Aldrich Chemical Company, Inc. and used without further purification. The buffers used here were the same as those used before (Huang & Cantor, 1975). Details of the preparation of ribosomes, 50 S and 30 S subunits, 16 S rRNA, batch eluted proteins and purification of individual 30 S ribosomal proteins have been described in the preceding paper (Huang & Cantor, 1975). (b) Synthesis qf [ZH]I-AEDANS The procedure described for unlabeled I-AEDANS by Hudson (1970) was followed except that the preparation was scaled down. all-labeled iodoacetic acid, obtained from New England Nuclear (spec. act. 377 mCi/mmol) was diluted with iodoacetie acid to a spee. act. of 4.0 mCi]mmol. I n one typical preparation 0.40 mmol of the diluted all-labeled iodoacetic acid was allowed to react with 0.48 mmol p-nitrophenol. The yield of p-nitrophenyliodoacetate was 65~o. This was reacted with 0.167 mmol 1,5-ethylene diamine naphthyl sulfonate to give all-labeled I-AEDANS at a yield of 70%. The identity of the product was confirmed by its spectral properties and thin-layer chromatographic behavior. The spec. act. of the all-labeled I-AEDA_NS, 3"8 mCi/mmol, was calculated from the radioactivity contained in a sample of known weight or of known ultraviolet absorbance. Both methods gave essentially the same result, suggesting that the compound obtained was reasonably pure. (c) _~luorescent conjuflat~ of individual 30 S proteins Purified individual 30 S ribosomal proteins were dissolved in 6 ~t-urea-0.30 ~-KC1 before reaction with fluorescent dye. 1 vol. of protein solution plus 0-5 vol. of 6 ~-urea in 0.5 Msodium carbonate (pH 9.5, 9.9 or 11.0) was mixed with about 100 M excess of dyes. t Abbreviations used: FITC, fluoreseein isothioeyanate; I-AEDANS, iV-iodoacetyl.N'-(5sulfonic-l-naphthyl)ethylene diamine; TP30, a mixture of all 30 S ribosomal proteins; mixes A, B, C, D are sets of hateh-eluted proteins; their preparation is described by Huang & Cantor (1975). Proteins S16 and S17 were always used as a mixture, called S16]17.

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Reactions with F I T C were carried o u t a t 0°C w i t k constant stirring for 5 to 20 min using Celite-adsorbed d y e as described previously (Huang & Cantor, 1972). I - A E D A N S labeling was done a t room t e m p e r a t u r e in t h e d a r k for 6 h or longer without Celite. U n r e a e t e d d y e was removed b y Sephadex G25 c h r o m a t o g r a p h y (carried out in the d a r k for I - A E D A N S samples) followed b y extensive dialysis against T R I buffer (see preceding paper) containing 2 ~-urea. To estimate the n u m b e r of dye molecules per protein molecule, the following assumptions were made. The molar extinction coefficient of b o u n d F I T C a t 495 n m has been assumed to be 4-26× 104 (Tengerdy & Chang, 1966}. See section (h), below for a discussion o f this choice. The molar extinction coefficient of I . A E D A N S a t 336 n m in w a t e r is 6.1 × 103 (Hudson, 1970), t h e value also used to determine the specific a c t i v i t y of 3H-labeled I - A E D A N S . The molecular weights of each of the purified 30 S proteins are those reported b y Voynow & K u r l a n d (1971). Protein concentration was measured b y the m e t h o d of Lowry e$ al. (1951 ), using crystalline bovine serum albumin as a s t a n d a r d . The recovery of the labeled proteins was a b o u t 50~/o. The m a i n loss was due to discarding n o n - p e a k fractions from Sephadex chromatography. Other loss is p r o b a b l y due to precipitation after labeling. W e found t h a t labeled proteins are less soluble t h a n nonlabeled ones. Sometimes as much as 4 K-urea was a d d e d to T R I buffer in order to keep labeled proteins in solution, particularly those with m a n y dyes per molecule. After one Sephadex gel filtration a n d extensive dialysis, usually a b o u t 10% of the t o t a l dye remaining with proteins was n o t covalently attached. This was removed b y one more gel filtration. Afterwards, all the remaining d y e a p p e a r e d to be covalently b o u n d to protein. The reproducibility of labeling was good with some proteins, a n d poor with some others. W e have no explanation for this. I n t h e ease of FITC-labeling, t h e p H of t h e reaction mixture a n d duration of the reaction needed to ensure high extents o f reaction correlate somewhat with the isoelectric points of the proteins (Kaltschmidt, 1971). A higher a n d longer reaction time was needed for proteins with higher isoelectric points. This is n o t true for I-AEDANS-labeling. F I T C can be expected to react with amino groups (Edman, 1956} a n d it is reasonable t h a t elevated p H should facilitate labeling. I - A E D A N S has been r e p o r t e d to be a s u l p h y d r y l reagent a t n e u t r a l p H (Hudson, 1970}. However, t h e I - A E D A N S r e a c t i v i t y of purified cysteine containing 30 S proteins (Graven e$ al., 1969) was low a t p H 7.7. $4 was more reactive t h a n others, y e t a 17-h incubation still yielded only 0.56 I - A E D A N S molecules per protein. Prolonging the reaction time up to 48 h did n o t increase the extent of reaction. Reduction of $4 and $8 with dithiothreitol according to the conditions described b y Slobin (1971) also did n o t improve the labeling. Those proteins containing no eysteine also reacted slightly w i t h I - A E D A ~ S a t p H 7-7, implying t h a t t h e d y e m i g h t react with amino groups. This was exploited in all further experiments b y using I - A E D A N S a t more alkaline p H values. A m a r k e d increase in e x t e n t of reaction was observed. (d) Reassembly of labeled proteins into 30 S subunit~ 20 to 24 A260 units of 16 S R N A were incubated in 8.0 to 9.0 ml reconstitution buffer a t 40°C for 40 rain w i t h 2.0 equivalents of labeled proteins a n d either j u s t those proteins necessary for binding (Mizushima & Nomura, 1970) or else m o s t of the other 30 S proteins (in a m i x t u r e of purified proteins a n d batch-eluted proteins). After t h a t , 1-5 equivalents of total 30 S proteins in reconstitution buffer were a d d e d to supply a n y missing proteins which, owing to scarcity of m a t e r i a l could n o t be a d d e d in purified form. The incubation was continued for another 60 rain. The completed particles were recovered b y centrifugation and dissolved in Tris/Mg/NH 4 I buffer. The incorporation of fluorescent dyes into the S0 S subunit was c o m p u t e d assnrn~ngt h e particle has a molecular weight of 0-9)< 108 (]:[~n e~ a/., 1969}. Ribosome concentration could be determined from t h e absorbance a t 260 n m (Azmflml ~ 15"0) since the contribution of t h e dyes to the t o t a l absorbance a t this wavelength is absolutely negligible. The a m o u n t of F I T C p e r S0 S is obtained from the absorbance a t 495 nm, the F I T C m a x i m u m . A t 1"5 m g 30 S/ml (the concentration used for most experiments), one equivalent of F I T C will have ,44g 8 a b o u t 0.07. Therefore a 0.1 slide wire on a Cary 15 spectrep h o t o m e t e r was used for these measurements. A separate SO S sample containing no

446

K . - H . H U A N G , R. H. F A I R C L O U G H AND C. R. C A N T O R

fluorescent dye was used as a b l a n k to correct for scattering. A t 495 l u n this contributed about 0.002 units to the absorbance. The ~raax of I-AEDA_NS is at much shorter wavelength t h a n F I T C a n d the ema~ is almost a n order of magnitude smaller. Thus it is impractical to make a n accurate determination of I-AEI)A_NS incorporation into the 30 S particle b y absorption measurements. I n s t e a d this was determined b y liquid scintillation counting of the 3H-labeled I - A E D A N S in I ~ n a r d ' s (1957) solution. Samples were counted for 70 min, because of the low specific activity of the dye. (e) Sedimentation and activity of rea~s~nSled particles The sedimentation rate a n d functional activity of reconstituted particles containing FITC-labeled and/or I-AEDANS-labeled proteins were examined. The recovered 30 S particles in TrisflYig/NH4 1 buffer were incubated at 37°C for 30 m i n to remove aggregates before sucrose-gradient centrifugation. A Jail[uracil-labeled 50 S and 30 S mixture was used as a marker. For activity assays, samples (65 ~1 containing 0.15 to 0.20 A~6o units of reconstituted particles) were t a k e n from the reconstitution mixture a n d the activity of reconstituted particles i n poly(U)-directed [z4C]phenylalanine incorporation (Nirenberg, 1963; ]~osokawa et al., 1966) was determined. A control particle was prepared b y replacing fluorescent-labeled proteins with corresponding non-labeled ones. (f) Energy transfer e~ficiencies The samples used for this purpose were all freshly prepared. They were clarified b y low speed eentrifugation, followed b y incubation at 37°C for 30 rain. Then the samples were kept at 0°C a n d fluorescence measurements were carried out within 24 h in most eases. To determine the transfer efficiency, E, between a donor-labeled protein a n d a n accepterlabeled protein i n a reconstituted 30 S subunit, three reconstituted particles were examined spectrofluorimetrieally. 1. Reconstituted 30 S subunits with donor-labeled protein. 2. Reconstituted 30 S subunits with donor-labeled a n d accepter-labeled proteins. 3. Reconstituted 30 S subunits with unlabeled protein. The three samples contained roughly the same A280 of ribosomes. Each sample was placed i n one of a matched set of 1.0 em × 1.0 cm fluorescence cells a n d the 3 cells were placed in a 4-sample cell block of a Perkin Elmer MPF-2A spectrofluorimeter thermostatically controlled a t 21°C. After a preliminary spectral characterization, the excitation monoehrometer was set a t a wavelength absorbed significantly b y the donor, a n d the emission monoehrometer was set at a wavelength rich in signal from donor a n d devoid of signal from accepter. The emission a n d excitation slits were routinely set at 5 n m bandpass. The resulting signals from each of the 3 samples will be denoted, respectively: PD; PDa; F3o. Fractions of the first 2 samples were also examined b y K i n a r d ' s (1957) solution liquid scintillation counting for the presence of 3H-labeled I-AEDANS donor in the samples. We denote Dcpm as the ets]min i n 30 S with donor-labeled protein a n d DAopm as the c~s/rnin i n 30 S with donorlabeled a n d accepter-labeled proteins. F r o m the spectrofluorimetrie a n d scintillation counting measurements, a n observed transfer efficiency, E, was computed using the following equation:

E

~-

1

(PoA (.Fo -

-- F30) ~3o)

Dcpm ' D--'Z~,~"

Generally, the reconstituted 30 S samples examined speetrofluorimetrieally contained 5 to 6 A=60 units of 30 S. Occasionally because of variation i n the incorporation of dyes into 30 S subunits, higher or lower concentrations of 30 S were used. The unlabeled 30 S samples always contained 4 to 7 A26o units. Whenever both the labeled a n d unlabeled 30 S were in the range of 4 to 7 A2eo, differences in scattering resulting from concentration differences were negligible. I f the labeled and unlabeled 30 S differed i n A26o more substantially, the unlabeled 30 S signal was corrected for this difference b y assuming t h a t the 30 S scattering was directly proportional to A=eo.

ENERGY

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447

F o r every determination of E two sets of excitation a n d emission wavelengths were used. These were t y p i c a l l y : ~ x = 360 nm, hem = 470 n m a n d ~ x = 350 nm, hem ----460 nm. The E values determined from these two trials agreed closely with one another, frequently giving the same value of E a n d rarely giving values of E which differed b y more t h a n 0.02. Occasionally, determinations of E were m a d e several hours a p a r t on the same samples with different slit-widths a n d amplifications, and the various values of E rarely differed b y more t h a n 0.02. I n addition to monitoring E, the transfer efficiency b y s t e a d y - s t a t e donor quenching as described previously, we a t t e m p t e d to monitor E b y d y n a m i c quenching of donor lifetimes in a p h o t o n counter a n d also b y s t e a d y - s t a t e sensitized emission of the accepter fluorescence. The lifetime d a t a was collected using the first two samples described previously. These samples were excited b y the spectral o u t p u t of a free-running air s p a r k l a m p filtered t h r o u g h a Coming 7-60 or 7-51 filter. The emission collected a t 90 ° to excitation was passed through a J a r r e l Ash ¼ meter monoehrometer set a t 470 nm. The d a t a collection rate was routinely <0-01 of the l a m p s p a r k rate. The d a t a collected have been difficult to interpret quantit a t i v e l y owing to our i n a b i l i t y to resolve the lifetimes a n d amplitudes of the shorter lifetime components of the samples with a c c e p t e r present. F o r samples showing substantial transfer efficiency b y s t e a d y - s t a t e donor quenching, the long lifetime component a m p l i t u d e is smaller for t h e d o n o r - a c c e p t e r sample t h a n for the donor sample. This is consistent with t h e s t e a d y - s t a t e quenching. Unfortunately, t h e heterogeneity of the population of species with donor a n d accepter present simultaneously makes q u a n t i t a t i o n of E b y observing the shortening of donor lifetime experimentally impractical. The a t t e m p t s to monitor E b y sensitized emission from the accepter are complicated b y the presence of donor fluorescence a t a n y wavelength one might choose to monitor t h e accepter fluorescence. Also the determination requires a fourth sample above a n d b e y o n d w h a t is required for measuring E b y steady-state donor quenching, n a m e l y : reconstituted 30 S subunits with accepter-labeled protein. After seeing t h a t sensitized emission a n d donor quenching gave roughly equivalent results on a few samples, we proceeded to msasure E b y s t e a d y - s t a t e donor quenching alone. (g) Measurement of quantum eO~clency of donor labeled 30 S subunits The q u a n t u m efficiency of the donor, I - A E D A N S , a t t a c h e d to different ribosomal proteins in reconstituted 30 S subunits was determined using quinine bisulfate in 0-1 l¢HaSO4 as a reference with ~(QS) = 0-70 (Scott e t a / . , 1970). Routinely the uncorrected emission spectra obtained using 360 n m excitation and 5 n m excitation and emission sllt bandpasses were recorded for 3 samples: 1. P r o t e i n - A E D A N S in reconstituted 30 S. 2. Reconstituted 30 S. 3. Quinine bisulfate in 0.1-NH2SOd. The spectrum of the second sample was a d j u s t e d for a n y A2e0 difference between sample 1 a n d sample 2. This a d j u s t e d spectrum was then subtxacted from the spectrum of 1 to give the spectrum of b o u n d I - A E D A N S resolved from the scattered light signal. The spectra of bound I - A E D A N S a n d of quinine bisulfate were then corrected for wavelength dependent emission sensitivity of the fluorescence detection system of the Perkin E l m e r MPF-2A. The 360 n m absorbanee of the I - A E D A N S - p r o t e i n - r e c o n s t i t u t e d 30 S was calculated from the concentration of b o u n d I - A E D A N S using a n extinction coefficient of 6.8 × 108 M - I c m -1 (Hudson, 1970). The concentration of b o u n d I - A E D A N S in t h e sample was determ i n e d b y K i n a r d ' s (1957) solution liquid scintillation counting. The 360 n m absorbance of t h e quinine bisulfate sample was measured directly using t h e 0 to 0.1 range of a Cary 15 spectrophotometer. The corrected spectra a n d t h e 360 n m absorbances were t h e n used to compute t h e q u a n t u m efficiency of t h e b o u n d I - A E D A N S b y s t a n d a r d methods (Parker, 1968). The required numerical integrations were performed b y Simpson's rule on an I B M 360-91. (h) Calculation of Re R0, the distance between a single donor a n d a single accepter a t which the singleC~singlet transfer efficiency is 0.50, depends upon: the spectral overlap, J , of the donor

448

K . - H . H U A N G , R. H. F A I R C L O U G H AND C. R. C A N T O R

emission a n d aeeeptor absorption; the geometric orientation between the donor a n d acccpfor, K~, the q u a n t u m efficiency of the donor i n the absence of transfer, ~0, a n d the refractive index, n, of the m e d i u m between donor and acccptor. Explicitly:

RoS=[ $'79x10-SemMnm-4Ae]

~-i ~oJ

in which J = ~Fv(A)EA(~)A2d~ in units of cm -1 ~ - 1 n m 4. SFD(A)A-SdA In the above equation 2'D is the corrected emission intensity of the donor at wavelength A, and EA(A) is the extinction of accepter. The above equations are set up to use absorption data and fiuoreseence data expressed in wavelength as routinely obtained in our laboratory. Other expressions of R o have been quoted (Stryer, 1968) which use raw data in wavenumbers. Ro was calculated for each I-AEDANS-labeled protein. The spectral characteristics of the various F I T C - p r o t e i n conjugates were quite similar so t h a t the absorption properties of all conjugates were approximated b y a single FITC--protein conjugate. There is an ambiguity in the proper choice of em~x for FITC-derivatized proteins. Presently available quantities of F I T C - 3 0 S precluded our making a determination of ~raax. Therefore, we have chosen the value 4.26× 104 M- I cm -~ (Tengerdy & Chang, 1966). This value has fit best in the past with energy transfer measurements between randomly labeled proteins in simple complexes (Gennis et al., 1972; Gennls & Cantor, unpublished results). A n alternative choice of ~max, 6"36 × 104 M-1 em -1 (Mercola et al., 1972) adjusted for p H from 8max of FITC-labeled insulin, would decrease all of the labeling stoichiometries we report b y a factor of 0-67. This would generally decrease center to center R/Ro b y 0.1 to 0-2 depending on the protein pair. However, the change i n emax would increase Ro from 42.5/1 to 45.5~ partially compensating for the decrease in t~/Ro. Overall, the effect would be to lower the center to center distances reported in Table 13 b y 10% at the most. Since this would make a few of the measured distances unreasonably small, we have chosen for the time being to use the smaller reported value of 8max. The spectral overlap integral, J, was computed b y Simpson's rule integration on a n IBM 360-91 computer. The orientation factor, K2, was assigned a value of 0.666 consistent with dynamic averaging, and the refractive index was set at 1.4. The values for R 0 of the various I-AEDANS-labeled protein samples ranged from 40 A to 45 ~ with a n average value of 42.5 ~ . A few representative values are: Donor

~o

I-AEDANS-S20 I-AEDANS-S16/17 I-AEDANS-S19 I-AEDANS-S13 I-AEDANS-S15

0.53 0.30 0.45 0.46 0.33

j(~-icm-lnm

~) x 10 -15

0.941 0.954 0.976 1.040 0-941

R oCA) 44.4 40,4 43.4 43.8 40.9

(i) Interpretation of energy transfer reauZts I n a simple system, containing only 2 chromophores in perfect 1 : 1 steiehiometry and fixed positions, the energy transfer efficiency immediately yields the distance between them. As shown b y FSrster (1965):

E =/1o6/(/¢o 6 + R6). Here E is the measured efficiency, R is the interchromophore distance a n d R0 is the characteristic transfer distance. R0 can be calculated for a given pair of chromophores as described above. A major source of u n c e r t a i n t y i n the calculation of Ro, a n d thus the determination of R is the relative orientation of donor a n d acceptor. This appears as a geometric factor, 0 ~ K2 _~ 4. Ro (and thus R) depend on (~u)1/6. I f the chromophores are t u m b l i n g rapidly a n d have r a n d o m orientation, K2 ----~. I f they are r a n d o m b u t rigid, K2 = 0"475 (Maksimov & Rozman~ 1961). Usual practice is to guess one.of these values

ENERGY TRANSFER IN RIBOSOMAL PROTEINS

449

for K~ a n d t r y to demonstrate t h a t the results are n o t as sensitive as t h a t to the postulated value. This is done either b y demonstrating chromophore motion or b y showing t h a t altered choices of positions of dyes yield similar results. As long as x2 does n o t have a n extremely small value, the distances computed from energy transfer are probably affected b y the choice of K2 b y no more t h a n 20~o. Since we are interested in qualitative results here, it will be sufficient to assign K2 a value of ~ a n d accept this uncertainty. I f it were justified, more elaborate methods exist for assessing a n d sometimes reducing the u n c e r t a i n t y introduced b y ~2 (Dale & Eisinger, 1974}. However, it is highly likely that some of the assumptions we m u s t make below outweigh the importance of a particular choice of ~ . The likely rotational freedom of dyes attached to lysine side chains a n d the insensitivity of the results to dye p e r m u t a t i o n argue t h a t ~ is a reasonably accurate choice for K2. Two extreme models have been used to analyze measured energy transfer efficieneies in terms of specific distances. Both are serious approximations and, as will be shown later, the numerical results of both methods are quite similar b u t the distances derived have different meanings. Model 1. I n the absence of further information about the system, we assume t h a t dyes are located at r a n d o m on the surface of spherical proteins. I n s t e a d of a fixed n u m b e r of donors and accepters, we allow for a distribution of stelchiometries. As long as the probability for donor-donor energy transfer is small, the measured energy transfer efficiency, E, will be a function only of the n u m b e r of acceptors, the radius of the proteins a n d the distance between t h e m (Gennis & Cantor, 1972).

I n ~his equation, f i is the fraction of systems containing i accepters, J~ is the resulting energy transfer efficiency for t h a t stoichiometry. E~ can be calculated from the geometry of the system a n d knowledge of R0 (Gennis & Cantor, 1972). The radius of each particular protein is estimated from its molecular weight and typical values for partial specific volumes. T h e n a grid of values of/~ is calculated as a function of the distance between the centers of the proteins a n d the distribution of accepter stoichiometries, f~. This is compared with measured values of the efficiency as a function of the n u m b e r of acceptors. Previous work has shown t h a t assuming a Poisson distribution of acceptors yields reliable values for the distances between proteins in simple oligomeric systems (Gennis et al., 1972; Gennis & Cantor, unpublished results). This is a convenient distribution since the f~ values are all uniquely specified b y the mean n u m b e r of accepters per protein. Energy transfer efficieneles calculated using the Poisson distribution are shown in Figure 3. Keep in m i n d t h a t results are the distances between the centers of the proteins. F o r reassembled 30 S samples there is a n a m b i g u i t y in the determination of the m e a n n u m b e r of acceptors. The reassembled particles contain a lower n u m b e r of dye molecules per protein molecule t h a n the pure labeled proteins. Two different assumptions might be consistent with our state of knowledge a b o u t the system. One extreme is to assume t h a t the reassembled ribosome contains a Poisson distribution of dye with the mean as measured i n the particle. This has been used for all results shown in this paper. The other is to assume t h a t the reassembled particle contains a mixture of unlabeled proteins a n d proteins with dyes distributed according to a Poisson distribution, with the mean n u m b e r of dyes per protein found in the original pure labeled sample. I n this case, the measured transfer efficieneies must be adjusted to account for the fraction of particles with no chance of containing a labeled protein. These cannot participate in energy transfer. The correct efficiency is the measured value divided b y the fractional equivalent of incorporated labeled protein. This can now be used, in conjunction with the m e a n n u m b e r of acceptors per original labeled protein, to compute distances. Calculations based on both sets of Poisson assumptions were done. I f the distance between two proteins is large, as, for example, $4-S 19 or $4-S 15, it makes no difference which statistical scheme is chosen. I f the distance is short, the distances computed using the second assumption are in m a n y instances too short to be realistic since they lead to overlapping proteins. Thus, this assumption was discarded. 31

450

K.-H. HUANG, R. H. F A I R C L O U G H AND C. R. CAI~TOR

Model 2. We assume a Poisson distribution of dye stoichiometries as above, but instead of allowing for random surface location, we assume that all dyes have reacted at one unique site. The rest of the data analysis proceeds as before except that the result derived now refers to the point to point distance between labeling sites on the two different proteins. Since the mean number of accepters incorporated into ribosomes is usually 1-0 or less, using a Poisson distribution is not very different from assuming a single reactive residue. However, statistically it is an admission of our ignorance of any details of the labeling reaction.

3. R e s u l t s Direct reaction of fluorescent dyes with intact ribosomes or subunits does not yield fluorescent derivatives selective enough to enable meaningful energy transfer experiments to be done (Huang & Cantor, 1972; Hsiung & Cantor, 1973). Thus it was decided to label individual purified ribosomal proteins and reassemble them back into 30 S particles. Previous work had indicated t h a t this was likely to be a successful way of generating specifically labeled active 30 S particles (Huang & Cantor, 1975; Held, Nomura, Gennis, Huang & Cantor, unpublished results). At the outset, a critical choice of strategy had to be made. I f unique fluorescent derivatives of particular amino acid residues of isolated proteins could be produced, subsequent energy transfer measurements on reassembled ribosomes would yield accurate distances between the labeled sites. Although this approach will clearly be the focus of future work, it was discarded in planning the present studies. The difficulty of preparing and characterizing numerous uniquely labeled proteins seemed formidable. Furthermore, without additional structural and sequence information on ribosomal proteins, it would be difficult to compare the results of such energy transfer experiments with any other existing structure information, l~or example, if one knew that two fixed sites on a pair of ribosomal proteins were 60 ~ apart, it still leaves enormous ambiguity about the relative proximity of the proteins considered as a whole. T h e y might still be in contact, with both labeled sites located far away from the contact surface. The approach chosen was to label individual purified proteins as non-specifically as possible. I f successful, this produces a distribution of dyes on the surface of each protein. Assuming a Poisson distribution of labeling stoiehiometry and random surface location on spherical proteins, energy transfer measurements between pairs of such labeled proteins can be analyzed to yield the distance between the centers of the two proteins (Gennis & Cantor, 1972). Successful applications of this approach have already been accomplished in a variety of simple protein complexes (Gennis et al., 1972; Gennis & Cantor, unpublished results). (a) Reaction of fluorescent dyes with ribosomal proteins FITC and I-AEDANS were coupled covalently to purified individual 30 S ribosomal proteins as described in Materials and Methods. Rather alkaline conditions, p H 9-5 to 11-0, were used to a t t e m p t to produce non-specific conjugates containing several copies of fluorescent dye. In most cases, this last objective was achieved successfully as shown by the results in Table 1. Increased incubation times were usually unsuccessful at raising the degree of labeling of the proteins. Protein conjugates with less t h a n one fluorescent dye per molecule were sometimes relabeled to raise the degree of dye substitution to acceptable levels and to conserve these valuable samples. These experimeats led us to the

ENERGY

TRANSFER

IN RIBOSOMAL PROTEINS

451

TXB~E 1

Extent of reaction of 30 S larotein8 with $'ITG or I - A E D A N S Protein

pH~f

$4 $5 $6 $7 $8 $9 Sll S13§ S14 S15 S16[17 S18 S19 $20

9.9 11.0 9.9 11.0 9.5 11.0 -11.0 9.5 11.0 11.0 9.5 11.0 9.5

FITC-Celite reaction Time Dyes/proteinS (rain) 10 10 10 5 5 5 -I0 6 5 5 10 20 10

2.2-2.9 3.4 0.8-1.4 1.2 1.0 1.9 -2-0 2.3 2-0 1.5-1.8 1.0 1.0 1-8

pHt

I-AEDANS reaction Time Dyes[protein~ (h)

7.7 9.5

17 20

9.5 9.5 9.5 9.5 9-5 9.5 9.9 9-9 9.5 9.9 9.5

22 19 15 6 22 6 19 17 6 15 22

0.50-0.66 2.20 2.86 2.60 3.10 1.42-1.64 1.24-2.16 2.28 3.06 2.34 1.07 2.75 3.23

Carbonate buffer was used except for $4 (pH 7.7) where phosphate buffer was used. :~The data were obtained after subjecting labeled pro~ins to one Sephadex column and extensive dialysis. The true covalent attachment could be as much as 10% less. §Molecular weight used is that reported by Garrett & Wittmann (1973).

curious finding t h a t , for $4 p r o t e i n , p r e - e x i s t i n g e o v a l e n t l y a t t a c h e d F I T C or I A E D A N S e n h a n c e d s u b s e q u e n t F I T C r e a c t i o n , b u t n o t I - A E D A N S reaction. P r e e x i s t i n g F I T C on S19 also e n h a n c e d f u r t h e r F I T C . l a b e l i n g . S o m e of t h e s e results are p r e s e n t e d in T a b l e 2. A h i g h degree o f d y e s u b s t i t u t i o n facilitates e n e r g y t r a n s f e r m e a s u r e m e n t s . I n t e r p r e t a t i o n o f t h e m e a s u r e m e n t s is m a i n l y s e n s i t i v e t o t h a t f r a c t i o n o f t h e s a m p l e c o n t a i n i n g n o e n e r g y a c c e p t o r . A v e r a g e n u m b e r s o f a c c e p t o r s p e r p r o t e i n o f t w o or m o r e r e d u c e this f r a c t i o n t o m a n a g e a b l e levels. T h e use o f e v e n m o r e h e a v i l y l a b e l e d

TABLE 2

Enhancement of EITO-labeling by pre.existing dye on Trotein molecules

Sampler

$4 $4 I-AEDANS-S4 I-AEDANS-S4 FITC-S4 S 19 FITC-S19

Prior dye[protein

None None 0-20 I-AEDANS 0-48 I-AEDANS 0.00 FITC None 1.0 FITC

pH

Reaction time (min)

FITC reaction/proteln

9-5 9.5 9.5

10 20 5

1.9 1.7 2-4

9.5

10

4.8

9.5 11.0 U.0

6 20 20

3.4-4.0 1.0 :> 2.0

? The labeling procedures were as described in Materials and Methods.

452

K . - H . H U A N G , R. H . F A I R C L O U G H

A N D C. R. C A N T O R

samples would offer additional advantages in the fluorescence measurements (Gennis & Cantor, 1972) but would surely decrease the chance of successful reassembly of the proteins into active 30 S particles. (b) Incorporation of labeled proteins into 30 S particles The feasibility of singlet energy transfer experiments on ribosomes is contingent on the ability to incorporate fluorescent dyes specifically into biologically active particles. The strategy that has been used to accomplish this is reassembly of individual or pairs of fluorescent-labeled 30 S ribosomal proteins. With two different dyes and 21 proteins, there are 420 double-labeled particles that can be constructed. Only a small fraction of these have thus far been attempted. Several criteria were used to select the test cases for these pilot studies. Where possible we wanted to measure energy transfer between pairs of proteins for which other information on proximity already existed. This would permit an evaluation of the reliability of our results. Differences in the availability of individual purified proteins and the ease of preparing fluorescent conjugates containing one or more dyes was another important consideration. The pair of dyes we have chosen is mostly suitable for measuring the quenching of the donor, I-AEDANS, by the acceptor, ~'ITC (v/de infra). For such measurements the fraction of particles that contain donor is relatively unimportant. However the quality of the data is strongly dependent on good incorporation of the acceptor. Where possible, acceptors were placed on proteins known to have unit stoichiometry (Garrett & Wittmann, 1973; Kurland, 1972). Alternatively, if it were known that reassembly of protein A depended on the pre-existence of protein B, B would contain the acceptor and A the donor. In general, 1-0 equivalent of 16 S rRNA was mixed with 2.0 equivalents of fluorescent-labeled protein plus at least all those unlabeled proteins known to be required for its binding (Miznshima & Nomura, 1970). After incubation, 1.5 equivalents of TP30 were added to the system and incubation was continued. Some typical results are shown in Tables 3 to 6. Final addition of TP30 was necessary because, in our hands, reassembly m~tures in which all proteins were purified were not very efficient at fielding 30 S particles (Huang & Cantor, 1975). Furthermore, the use of all purified proteins in every experiment is inconsistent with available quantities. The use of TP30 has one serious shortcoming. TP30 contains unlabeled copies of the same proteins that pre-exist in the assembled particles as fluorescent derivatives. If exchange can occur, the degree of final incorporation of labeled proteins will be diminished. The number of fluorescent dyes per 30 S particle is determined for FITC by absorbance and for [3H]I-AEDANS by incorporation of radioactivity as described in Materials and Methods. There is no ambiguity in the analysis of double-labeled particles. There is, however, a serious ambiguity in the interpretation of the measured extent of incorporation of fluorescent dyes. If a protein with an average of n dyes per molecule incorporated into 30 S particles with unit stoichiometry and no preference for lightly or heavily labeled components, one would expect 30 S subunits containing an average of n dyes. As shown in Tables 3 to 6, this is never observed. Reassembled particles always contain fewer dyes than expected. The probable reasons for this are preferential selection of lightly labeled proteins during initial incubation, the lack of stoiehiometric binding, and subsequent exchange with unlabeled proteins after TP30 addition, as cited above. The conditions used for assembly permit the system to select for particular labeled protein derivatives capable of successful incorporation into 30 S

E N E R G Y T R A N S F E R IN RIBOSOMAL P R O T E I N S

453

TABLE 3

Effect of dye content on the incorpora$ion of labeled proteins into 30 S particle~ Labeled protein

FITC per protein

I-AEDANS per

$4

1.9

--

3.4

--

4.1

--

--

0.68 2.65 1.22 2.26 1.24 2.16 0.94 3-23

$8

- -

$9 • S13

--- - -

$20

- - -

protein

Other proteins present in the formation of the initial RNA-protein complex

Dyes] ribosome

~°~

I-AEDANS-SI9 and most other 30 S proteins~ I-AEDA_NS-S18 and most other 30 S proteins~ I-AEDANS-S20 and most other 30 S proteins~ most other 30 S proteins~ most other 30 S proteins~ most other 30 S proteins~ most other 30 S proteins~ $4, $8, $20 $4, $8, $20 $4, $8 $4, $8

0.40

0'21

1.56

0.46

1-27

0-31

0.33 0.29 0.59 0.41 0.40 0.28 0.18 0-29

0.49 0.11 0.48 0.18 0.32 0.13 0.19 0.09

A combination of batch-eluted proteins and purified proteins. S1 and $2 were absent in all eases. If the labeled protein is one of the proteins in mix A (Huang & Cantor, 1975), then $3 was missing as well; if it is one of the proteins in mix B, then both S12 and $21 were net included. :~Equivalents of labeled protein per 30 S particle if the isolated 30 S contain a mixture of unla~oeled protein and protein labeled with the same average dyes/protein as the starting labeled protein sample. particles. Thus it must be considered highly probable t h a t at least some reassembled proteins will in fact be specific fluorescent derivatives rather than randomly labeled samples. We have not yet carried out the large number of peptide analyses needed to examine this. Instead, as a crude measure of the efficiency of incorporation of fluorescent-labeled proteins, one can calculate the equivalent number of copies assembled per particle, F., assuming t h a t each 30 S particle contains either a protein with the same average number of dyes per molecule as the starting mixture or else a protein with no dyes (see Table 3). Fe can be taken as an estimate of the minimum fraction of 30 S particles containing the original protein with an average of one or more dyes. This assumes t h a t any preferences in assembly will favor lightly labeled proteins. The maximum fraction of particles that could contain a dye is either the number of dyes per ribosome, or 1.0, whichever is less. Generally, the range between minimum and maximum is less t h a n a factor of three. Increasing the extent of labeling of an individual protein usually reduces the average efficiency of reassembly. Sometimes the use of more heavily labeled protein actually leads to a smaller number of dyes incorporat~l into the reassembled particles. See the results shown for F I T C - S 4 in Table 3. Assembly of normal ribosomal proteins is dependent on or stimulated b y the presence of other 30 S proteins. The complex set of interrelationships which holds is summarized in the assembly map (MJzushima & Nomura, 1970; Held et aL, 1974) (see Fig. 4(b)). I t is reasonable to suppose t h a t the relatively poor efficiency of reassembly with fluorescent proteins results from a weakness of some of these interactions. This would facilitate loss of labeled protein at intermediate stages in reassembly b y exchange with unlabeled proteins. To examine this, the effects of proteins

454

K . - H . H U A N G , R. H. F A I R C L O U G H

A N D C. R. C A N T O R

T~LBLE 4

Influence of protein composition of initial RNA-~orotein incubation mixture on the incorporation of dye-labeled proteins into 30 S particle8 Labeled protein $4

FITC per protein

I-AEDANS per protein

Other proteins present in the formation of the initial RNA-protein complex

2.2 3.4 3.4 3.4 3-4 3-4

-------

--

2.16 2.16

None $8, I-AEDANS-S20 $7, $8, $20, I-AEDANS-S13 $5, $8, S10, I-AEDANS-S16/17 $8, S16/17, 820, I-A~DANS-SI5 I-AEDANS-SI8 and most other 30 S proteinst $4, $8, $20 $4, $8, $20 and most other 30 S proteinst None $8, S16/17, $20, FITC--S4 $4, $5, $8, $20, FITC-S16/17 84, 87, 88, 820, FITC-S19 $4, $5, $8, $20 most other 30 S proteins~ $4, $7, $8, $20 most other 30 S proteins~ $4, $7, $8, I-AEDANS-S20 I-AEDANS-S13 and most other 30 S proteLns~ 84, $8 84, $8 and most other 30 S protems~ $4, $7, $8, S16/17, I-AEDANS-S13 I-AEDANS-S13 and most other 30 S proteinst

S13

-

S15

---

-2.0 2-0

2-02 2.02 2-02 2-02 2-34 2.58 2.76 2-21 ---

-m

0-94 1.53

1.85 1.85

---

-

S16/17 S19

$20

-

-

----

-

Dyes/ ribosome

Fo~

0.51 0.92 1.16 1.19 1.43 1-56

0-23 0.27 0-34 0.35 0-45 0-46

0.28 0.45

0.13 0.21

0.34 0.77 0.85 0.79 0.35 0.72 0.55 0.91 0"40 0"54

0.17 0.38 0-42 0-39 0-15 0-28 0.20 0-41 0.20 0-27

0.18 0.57

0.19 0.37

0,39 0.56

0-21 0.30

t See footnotes to Table 3. o t h e r t h a n t h o s e k n o w n to be r e q u i r e d for a s s e m b l y o f a g i v e n c o m p o n e n t were m e a s u r e d . These r e s u l t s a r e s h o w n i n T a b l e 4. I n general, t h e a d d i t i o n o f m o r e t h a n a m i n i m a l c o m p l e m e n t o f 30 S p r o t e i n m a r k e d l y e n h a n c e s r e a s s e m b l y o f a fluorescent component. F I T C - l a b e l e d $4 p r o t e i n b e h a v e s s o m e w h a t like a m u t a n t f o r m $ 4 - S U 6 d e s c r i b e d b y G r e e n & K u r l a n d (1971). T h a t f o r m b o u n d m o r e w e a k l y t o 16 S R N A t h a n w i l d t y p e . T h e b i n d i n g was e n h a n c e d b y a d d i t i o n o f $7, $8, a n d $20. A d d i t i o n o f t h e s e a n d o t h e r p r o t e i n s m a r k e d l y s t i m u l a t e d r e t e n t i o n o f I~ITC-S4. T h e e x p e r i m e n t s w i t h fluorescent p r o t e i n s a r e n o t c o m p r e h e n s i v e e n o u g h t o afford a d e f i n i t i v e e x p l a n a t i o n o f t h e i n c r e a s e d efficiency, b u t t h e y a r e c o n s i s t e n t w i t h t h e h y p o t h e s i s t h a t e x c h a n g e o f l a b e l e d p r o t e i n s i n a 16 S r R N A p r o t e i n c o m p l e x is i n h i b i t e d once a d d i t i o n a l p r o t e i n s a r e b o u n d . As a p r a c t i c a l c o n s i d e r a t i o n , t h e e n h a n c e d e f c i e n c i e s g a i n e d b y t h e p r o c e d u r e s s h o w n in T a b l e 4 g r e a t l y assist fluorescence studies. The relative efeiencies of incorporation of I-AEDANS and FITC-labeled protein i n t o 30 S p a r t i c l e s a r e c o m p a r e d i n T a b l e 5. I n all cases w h e r e s a m p l e s o f e q u i v a l e n t n u m b e r s o f d y e s p e r p r o t e i n were p r e p a r e d , L A E D A N S - l a b e l e d p r o t e i n s s h o w e d s u b s t a n t i a n y h i g h e r p e r c e n t a g e s o f r e t a i n e d d y e i n r e a s s e m b l e d ribosomes. T h e

ENERGY

TRANSFER

IN RIBOSOMAL PROTEINS

455

TABL]~ 5

Relative incorporation of F I TC- and I-AED ANS-labeled proteins into 30 S partide8 Labeled protein S13

FITC per protein

I-AEDANS per protein

1.8

--

-

2.21

S18

1.05 -2.0

-

S19

-

-2.58 -1.07 --

-

2.21

-

Dyes/

-~e~

ribosome

I-AEDANS.S19 and most other 30 S proteinst FITC-S19 and most other 30 S proteinst I-AEDANS-S 19 and most other 30 S proteins~ most other 30 S proteins~f most other 30 S proteinst most other 30 S proteins~ I-A_EDANS-SI3 and most other 30 S proteins t FITC-S13 and most other 30 S proteinst

2-16

-

SI6/17

Other proteins present in the formation of the initial RNA-protein complex

0.32

0.18

0.52

0.24

0.38

0.17

0.72 0.17 0.55 0.54

0.28 0.16 0.51 0.27

0.82

0.37

See footnotes te Table 3.

TABL~ 6

Influence of FITC-labeled proteins on the incorporation of I.AEDANS.labeled protein into 30 S particles Labeled proteins used for reoonstitution T I-AEDANS-S18 I-AEDANS-S18 FITC-S6 I-AEDANS-S18 FITC-S4 I-AEDANS-S18 FITC-S15 I-AEDANS-S13 I-AEDANS-S13 FITC-S4 I-AEDANS-S13 FITC-S19

I-AEDANS/ I-A_EDANS/ protein ribosome

F.~tFITC/ I-AEDANS protein

FITC/ ribosome

F,$-FITC

1.07 1-07

0.55 0.55

0.51 0-51

-1.4

-0.22

-0-16

1.07

0.40

0.37

3.4

1-56

0.46

1.07

0.43

0.40

2.0

0.70

0-35

2.16 2.16

0.48 0.37

0.22 0-17

-3.4

-1.12

-0.33

2.16

0.54

0-25

2.0

0-54

0.27

Initial incubation of these particles with the 16 S rRNA was done in the presence of most other 30 S proteins. :~ See footnote to Table 3. s m a l l e r size o f t h e I - A E D A N S c h r o m o p h o r e p r o b a b l y r e s u l t s i n less p e r t u r b a t i o n o f p r o t e i n c o n t a c t s t h a n F I T C . A s a m p l e o f $4 c o n t a i n i n g 0-5 e q u i v a l e n t o f I - A E D A N S b o u n d t o t h e 16 S r R N A a l m o s t as well as has b e e n r e p o r t e d for u n l a b e l e d $4 (Mizus h i m a & N o m u r a , 1970). U n f o r t u n a t e l y F I T C - l a b e l e d p r o t e i n s w e r e n e v e r as cooperative. F o r e n e r g y t r a n s f e r e x p e r i m e n t s i t is n e c e s s a r y t o p r e p a r e ribosomes c o n t a i n i n g one proteir~ labeled w i t h I - A E D A N S a n d a n o t h e r laboled w i t h F I T C . T h e r e s u l t s i n

456

K.-H. HUANG,

R . H. F A I R C L O U G H

A N D C. R . C A N T O R

Table 6 demonstrate that such 30 S particles can be successfully constructed. The presence of proteins containing large numbers of FITC does inhibit somewhat the efficiency of incorporation of I-AEDANS-containing proteins. However the effects are not that large and the resulting particles still contain sufficient quantities of fluorescent dyes to be useful. (e) Proloerties of reassembled 30 S ribosomes containing fluorescent-labeled Trot•ins All single and double fluorescent-labeled particles prepared were examined by analytical sucrose-gradient centrifugation. Typical results are shown in Figure 1. In all cases a single 30 S band is seen which sediments essentially coincidentally with a native 30 S marker. No evidence is seen for shoulders on the band which would have indicated possible heterogeneity of structure or composition. From the efficiencies of dye incorporation into the particles shown in Figure 1, one can compute that at least 17% should contain both FITC and I-AEDANS-labeled proteins. If these doublelabeled particles had a seriously perturbed quaternary structure they might have had a different sedimentation rate. No evidence for this was found, which suggests that structure perturbations, if they exist at all, are too small to affect the overall size and shape of the particle.

i ! l ,~ I

0"8

Cal

3os

l,o~sl

t800

f-AEOANS-S~5

~

/~ F,TC-S.

I

•

1200 0.4

i .// i\ ,/

I

t

~

600

o•

I ~o-,J ~*-t .... "/

~\.

\ \, %-o-~-~:~-~r~

I

0

.E E

e~

;~I

0.4

FITC - S 4

i!/ 0.2

f

I

0

J

y

1

4

1200

•

600

!e~•-o_e.e.4..e.e

~

,.o/o-H-.--O~o/ o "°

t800 : c

1

o.

"°~O',o-o-o-9-o-o

12 Fraction no.

20

0

FIG. 1. Sucrose-gradient eentrifugation of reconstituted 30 S particles eontainingboth I-AEDANSand FITC-labeled proteins. The particles in Tris/Mg/NI-I4 1 buffer were heated at 37°C for 30 mln in order to remove any possible aggregates. [SH]uraciLlabeled 50 S and 30 S subunits were included as markers. They were layered on 5 ml of 5% to 20% sucrose solution in Tris/Mg/NH4 II, buffer then centrifuged at 45,000 revs/min for 2.5 h in a Spineo SWS0.1 rotor at 4°C. The fractions were then collected from the bottom of the tubes and analyzed for 260 nm absorbanee ( - - 0 - - 0 - - ) and radioactivity ( - - O - - O - - ) after precipitation with 10% trichloroacetic acid,

ENERGY TRANSFER IN RIBOSOMAL PROTEINS

457

Poly(U)-direeted polyphenylalanine synthesis was used to test the functional integrity of reassembled 30 S particles containing fluorescent-labeled proteins. Control particles prepared b y reassembly, b u t containing unlabeled proteins, were defined as 100% active for purposes of comparison. Initial experiments showed t h a t these controls had activities generally quite comparable to those prepared b y incubation of 16 S R N A with 2.5 equivalents of TP30 in the presence of 0.05 to 0.10 K-urea. The effect of I-AEDANS-labeled proteins on the activity of reassembled 30 S particles is shown in Table 7. Of those tested only I - A E D A N S - S 1 3 causes significant loss of polyphenylalanine synthesizing ability. I f all of the incorporated S13 were to contain 2.2 dyes per protein molecule one could interpret the results of Table 7 to imply t h a t

TABLE 7

Poly( U).directed poIyphenylalanine synthesis activity of 30 S particles containing I-AEDANS-labeled proteins I-AEDANS-laboIod protein

Dye]protein

Dye/ribosome

_F,t

Relative activity:~ (%)

$5 $9 S13 S15 S16[17 S18 S19 $20

1-50 2.26 2-16 2-02 2-58 1-07 2.21 1.53

0.15 0.41 0.45 0.81 0-72 0.55 0.91 0.57

0.10 0.18 0.21 0-40 0-28 0.51 0-41 0.37

99.9 98.8 77.4 100.5 110.0 99.2 96.7 108.3

t See footnote to Table 3. :~Assayed in triplicate. Particles were assayed right after reconstitution without purification. The activity of control 30 S particles, prepared exactly as those containing labeled proteins except that the labeled protein was replaced by corresponding unlabeled protein, was considered to be 100%.

all particles containing this labeled S13 protein are completely inactive. However, a much more likely situation is a distribution of particles incorporating labeled S13 with an average of less t h a n one dye per protein. I n this ease, a substantial fraction of the I - A E D A N S - S 1 3 combining particles would still have retained appreciable levels of activity. The activities of 30 S particles reassembled from one I-AEDANS and one FITClabeled protein are sho~qa in Table 8. These are somewhat lower t h a n the activity of particles containing only I-AEDANS, y e t the worst case still shows more t h a n 60% of the polyphenylalanine synthesizing ability of control particles. I t is clear t h a t activity is lowest for those particles with the largest F I T C content. I f one assumes t h a t there has been no selection during assembly for lightly labeled proteins, one could conclude from Table 8 t h a t all particles containing FITC-S4, -$8, and -S13 are inactive. But, as discussed above, it seems more plausible to take a statistical view in which ease the generally high activities of the sar~ples in Table 8 can be viewed with optimism. At present the safest conclusion is t h a t there is no compelling reason to associate a n y fluorescent derivative ~ t h more t h a n partial inactivation of function, I n conjunction

TABLE 8

$7 $4 $7 S13 S16/17 $4 $8 S16/17

t See footnotes to Table 3. See footnotes to Table 7.

$9 S19 S19 S19 S19 $20 $20 $20

Labeled proteins I-AEDANS FITC

2.26 2.21 2.21 2.21 2.21 1.53 1.53 1.53

I-AEDANS/ protein 1-2 1.9 1.2 1-8 1.1 3.7 1.0 1-I

FITC/ protein 0.29 0.82 0.73 1.82 0.77 0.50 0-47 0.47

I-AEDANS-pro~oin dye/ribosome 0-13 0.37 0.33 0.37 0-35 0.33 0-31 0-31

-~et 0-17 0.40 0.28 0.32 0.19 1"15 0-26 0-21

FITC-protein dye/ribosome

0.14 0.21 0.23 0-18 0.17 0.31 0.26 0.19

~',t

94.1 76.4 92.6 81.4 92.3 63.3 76.1 86.2

Relative activity~

Poly(U)-directed Tolyphenylalanine synthesis activity of 30 S particles containing both I-AEDANS- and FITC-labeled Troteins

¢Jt

ENERGY

TRANSFER IN R I B O S O M A L

PROTEINS

459

with the normal sedimentation properties of the labeled particles, it is not unreasonable to assume that whatever structural perturbations the dyes have introduced are minor. (d) Energy transfer measurements

The fluorescence of typical I-AEDANS and FITO-labeled reassembled 30 S ribosomes is shown in Figure 2. A broad range of wavelengths exists in which I-AEDA_NS fluorescence can be observed in the absence of any possible contamination from FITC emission. Thus this dye pair is ideal for donor quenching, the method used for all energy transfer measurement results shown in detail here. Observation of I-AEDA_NSsensitized FITC emission is possible because of the different absorption and emission intensity distributions of the two dyes. However, since I-AEDANS fluorescence overlaps the FITC emission at all wavelengths, data analysis is complex. In several eases where measurements were accurate enough to interpret quantitatively, sensitized emission gave good agreement with static donor quenching. The danger of relying mostly on donor quenching is the risk of certain artifacts which can mimic energy transfer. However, the low absorbance levels of our samples make most of such artifacts negligible. In principle one could use fluorescence lifetime data to confirm energy transfer found in static measurements. However, for heterogeneously labeled samples energy transfer leads to a distribution of lifetimes which defies ready analysis. These difficulties notwithstanding, the I-AEDANS-FITC system offers one substantial advantage. The excellent overlap of I-AEDANS emission with FITC absorption visible in Figure 2 and the high extinction coefficient of FITC affords an unusually high value for the characteristic transfer distance, R o (Wu & Stryer, 1972). This allows the detection of energy transfer between proteins separated by over 40 A. I

l

//~_

/

I

I

I-AEDANS _ _ / ~ |

~ -sd-3os

/

/

I

\

J~

I

FITC-S20

,k~/.~ ~ - - 3 o s

///i

/1\' I

!

I

I

\'~

I

// -

280

I

360

~

~

/

I

440 X (rim)

",

I

520

I

600

I

680

FIG. 2. Fluorescence excitation a n d emission spectra of 30 S ribosomes containing a single I - A E D A N S or FITC-labeled protein. To facilitate comparison all spectra have been normalized to the same m a x i m u m intensity. I n actuality the FITO intensities are m u c h larger t h a n I-AEDANS.

K . - H . H U A N G , R. H . F A I R C L O U G H AND C. R. C A N T O R

460

E n e r g y t r a n s f e r m e a s u r e m e n t s h a v e b e e n carried o u t o n 20 pairs of p r o t e i n s reassembled i n t o 30 S r i b o s o m a l s u b u n i t s . T h e results of these e x p e r i m e n t s are summ a r i z e d i n Tables 9 to 11. All transfer efficiencies were o b t a i n e d b y c o m p a r i n g t h e fluorescence of I - A E D A N S - l a b e l e d p r o t e i n reassembled i n parallel e x p e r i m e n t s i n t o two 30 S samples. One c o n t a i n e d n o o t h e r labeled proteins; t h e o t h e r c o n t a i n e d a single F I T C - l a b e l e d protein. Since t h e I - A E D A N S was t r i t i u m - l a b e l e d , t h e c o n c e n t r a t i o n of donoz i n b o t h samples is k n o w n u n a m b i g u o u s l y . T h u s r e l a t i v e q u a n t u m yields can b e c o m p u t e d easily. I n general, d a t a was corrected for a n y s c a t t e r i n g or fluorescence of t h e u n l a b e l e d ribosomal c o m p o n e n t s . Efficiencies were c o m p u t e d b y averaging d o n o r q u e n c h i n g a t two wavelengths. Some a s s u m p t i o n s a n d details of t h e calculations are g i v e n i n Materials a n d Methods. T h e r e p r o d u c i b i l i t y of replicate optical m e a s u r e m e n t s o n a g i v e n pair of samples is e x t r e m e l y good. T h e r e is little d e p e n d e n c e of t h e observed e n e r g y t r a n s f e r efficiency o n t h e w a v e l e n g t h s chosen for observation. T h e precision of m o s t of t h e results s h o w n i n t h e Tables is a b o u t 2 % energy transfer. F o r samples which c o n t a i n low a m o u n t s of T~BLE 9

Pairs of 30 S proteins exhibiting appreciable energy transfer in reassembled 30 S ribosomes Donor:

I-AEDANS- Dye/ Dye] Accepter: Dye/ Dye/ labeled protein ribosome FITC-labeled protein ribosome $4 $20 $20 S13 S13" S13 S13" S19 $20 S16]17" S19 S19" $19 S16/17 S16]17" S15 S15" S15 S16]17 S16/17" $9" $9" S13 S13" S19 S15

0.5 3.2 3.2 1.2 2.2 1-2 2-2 2-2 3.2 2.6 2.8 2.2 2.2 2-3 2-6 2-0 3.1 2.0 2-6 2.3 2.3 1.0 2-2 2-2 2.2 2.0

0-53 0.39 0.23 0.31 0.37 0-32 0.52 0.81 0.44 0.50 0-46 0-64 0.84 0.28 0-43 0.85 0-86 0.97 0.39 0.51 0.29 0.26 0.25 0.44 0.79 0.79

$20 $4 $4 $4 $4 S19 S19 S13 S16/17 $20 $4 $4 $4 $4 $4 $4 $4 $4 S15 S15 $7 $7 $20 $20 S15 S19

3-7 2.2 3.4 2.2 3.4 2.0 2-0 1-8 1.8 1.9 3.4 4.1 1.9 3-4 4-1 3-4 4.1 1.9 2.0 1.2 1.2 2.2 1-6 1.9 2.0 2-0

0.42 0.49 0.83 0.57 1.00 0.43 0.54 0.32 0.25 0.29 1.15 1.43 0.41 1.17 1.21 1.50 1.42 0.36 0.79 0.40 0-17 0.56 0.39 0.55 0.77 0.44

Transfer efficiency (%) (%/accepter) 15 13 25 15 27 15 12 6 24 15 7 8 3 18 32 8 16 3 16 8 10 30 14 2 0 7

36 27 30 26 27 35 22 17 96 52 6 6 8 15 26 5 11 8 20 20 59 54 36 4 0 16

30 S particles were prepared as follows: labeled proteins were allowed to form complexes with 16 S RNA in the presence of other unlabeled proteins required for their maximum binding as inferred from the assembly map (Mizushirna & Nemura, 1970; Held et al., 1974), or in the presence of most other 30 S proteins (particles indicated by *). At the end TP30 was added to form complete particles.

ENERGY

TRANSFER

461

IN RIBOSOMAL PROTEINS

TABLE 10

Examples where the extent of donor labeling strongly affected energy transfer in 30 S reassembled ribosomes Donor:

I-AEDANS- Dye/ Dye] Accepter: Dye] Dye/ labeled protein ribosome FITC-labeled protein ribosome $8 t S8t S18t S18 t S20t $20 S20J" S20

0-7 2-7 0-6 1-1 1-5 3.2 1.5 3-2

0-49 0-16 0-45 0.54 0.50 0.23 0.46 0.44

S 15 S15 $6 $6 $4 $4 S16/17 S16]17

1-2 2-0 1.4 1.4 4.1 3.4 1.1 1.8

0.34 0.55 0.39 0.21 1.28 0.91 0-29 0.25

Transfer ei~oiency (%) (%/accepter) -- 6 29 --4 10 0 25 6 24

-- 18 53 -- 10 48 0 27 21 96

t Samples prepared as described in the legend to Table 9. TABLE 11

Reassembled 30 S ribosomes that show little or no energy transfer Donor: I-AEDANSlabeled s18 s18 s13 $20 s19 s14

Dye/

Dye/

Accepter:

Dye/

Dye/

protein ribosome FITC-labeled protein ribosome 1.1 1.1 2.2 3.2 2.8 2.3

0.40 0.42 0.23 0.24 0.43 0-23

$4 S15 S16]17 S19 S16/17 S19

3.4 2.0 1.5 2.0 1.8 2.0

1.56 0-70 0.30 0-41 0.34 0.49

Transfer efficiency (%/accepter)

(%) 2 4 0 3 -4 2

1 6 0 7 - 12 4

I - A E D A N S , t h e l a r g e s t source o f e r r o r i n c o m p u t a t i o n o f t h e e n e r g y t r a n s f e r efficiency is t h e p r e c i s i o n i n d e t e r m i n i n g t h e r e l a t i v e L A E D A N S c o n c e n t r a t i o n s . A m o r e critical t e s t is t h e c o m p a r i s o n o f e n e r g y t r a n s f e r i n 30 S p a r t i c l e s r e a s s e m b l e d f r o m different p r e p a r a t i o n s o f t h e s a m e p a i r o f l a b e l e d p r o t e i n s . Since e n e r g y t r a n s f e r is a s e n s i t i v e f u n c t i o n o f t h e n u m b e r o f a c c e p t e r s , w e h a v e chosen t o c o m p a r e t h e e n e r g y t r a n s f e r p e r a c c e p t e r . T h i s is a r e a s o n a b l e y a r d s t i c k since a t t h e s e l o w levels o f l a b e l i n g t r a n s f e r s h o u l d b e r o u g h l y l i n e a r i n t h e a c c e p t e r c o n t e n t for r a n d o m l y l a b e l e d s a m p l e s (Gennis & C a n t o r , 1972). N u m e r o u s e x a m p l e s o f e x c e l l e n t a g r e e m e n t b e t w e e n different r e a s s e m b l e d s a m p l e s a r e s h o w n i n T a b l e 9. One case, I - A E D A N S - S I 3 t o F I T C - S 2 0 s h o w e d s u b s t a n t i a l e n e r g y t r a n s f e r in one s a m p l e b u t n o t i n a n o t h e r . I n cases like t h i s a n d o t h e r s to b e discussed l a t e r , t h e h i g h e r e n e r g y t r a n s f e r r e s u l t is m o r e l i k e l y t o b e correct. Since p r o t e i n c o n t a c t s a r e w e a k e n e d in d y e - l a b e l e d ribosomes, e x c h a n g e w i t h u n l a b e l e d p r o t e i n s s h o u l d b e m o s t l i k e l y in d o u b l e - l a b e l e d samples. This will r e d u c e t h e o b s e r v e d e n e r g y t r a n s f e r . F a i l u r e t o r e a s s e m b l e p r o p e r l y is a n o t h e r l i k e l y source o f errors. This is m o r e l i k e l y to increase a d i s t a n c e b e t w e e n a g i v e n p r o t e i n p a i r t h a n r e d u c e it. F o r p r o t e i n s w h i c h a r e i n t e r d e p e n d e n t in a s s e m b l y , i t is possible t h a t t h e a s s e m b l y of d o u b l e . l a b e l e d p a r t i c l e s i n w h i c h d y e s a r e close could

462

K.-H. H U A N G ,

R. H. F A I R C L O U G H

AND

C. R. C A N T O R

be impeded. This will again reduce energy transfer. With all of these possible drawbacks, itisgratifying that the general reproducibility of the resultsin Table 9 isso high. An important control in energy transfer experiments is to switch the identity of the dyes. In a strictlydefined system this permits the possible occurrence of fixed orientations of dyes to be examined (Beardsley & Cantor, 1970). Equivalent energy transfer with altered dyes is good evidence that the geometric factor, K 2, is not in a sensitive range. Therefore, where possible we reversed the positions of donor and aceeptor on protein pairs. Four examples of such experiments are shown in Table 9. Three show fairly good numerical agreement. One, S16/17-$20, demonstrates extremely effective energy transfer with both permutations of I-AEDANS and FITC although the actual numerical results differ substantially. The above results suggest not only the lack of preferred dye orientation but are also consistent with random labeling. However, four other pairs of proteins showed evidence of non-random labeling with LAEDAN8. These results are summarized in Table 10. In each case when a lightly labeled I-AEDANS sample was used, little energy transfer could be observed. In fact, some samples showed slight negative apparent energy transfer. This is presumably an experimental artifact. The most likely cause is errors in determination of the relative donor concentration in the single and double-labeled samples. Naturally these errors will be largest for lightly labeled samples. The use of higher donor concentrations produced much more substantial energy transfer. We believe these latter results are a more accurate representation of the state of the proteins in the ribosome. They will be used for further analysis and interpretation. It is likely that some of the discrepancies are due to selective labeling. 820 is involved in two of the four cases. Here the labeling even in the lightly derivatized samples is still substantial enough to eliminate artifacts as the likely cause. Instead it is reasonable to guess that $20 may have a unique reactive site which is occupied by the first I-AEDANS molecule added. The location of this site must be far away from 84, and close but not very close to 816/17. Subsequent I-AEDAN8 reaction sites on $20 lead to substantial energy transfer to acceptors on both proteins. Thus these must be located far away from the initial reaction site. I f this explanation is correct, the observed results can be rationalized providing that protein $20 is not spherical but instead is elongated within the 30 8 ribosome. For subsequent comparisons we shall use the higher transfer efficiencies for $20-$4 and S16/17-$20. This seems reasonable because these results are reproducible and also agree better with measurements between the same protein pairs but with the locations of I-AEDANS and FITC permuted. Not all samples showed significant levels of singlet energy transfer. A number of protein pairs shown in Table 11 exhibited between 0 and 7~/o efficiency per aceeptor. The most likely explanation is that these proteins are far apart in the 30 S particle. However, artifacts such as those discussed earlier have not yet been rigorously excluded. Therefore these results must be considered tentative. (e) Analysis of energy transfer result8 A summary of all of the energy transfer results obtained is given in Table 12. To facilitate comparison, all results are shown as per cent efficiency per aeeeptor. Qualitatively, such normalized efficiencies are inversely proportional to distances. Where multiple determinations of efficiency produced comparable results, average values are

ENERGY TRANSFER IN RIBOSOMAL PROTEINS

463

TABLE 12

Summary of average energy transfer results: relative ~roximities of 19rotei~,Tairs Very close

% E/acceptor

816/17-$20 $7-$9

74

$8-S15

58 53

$6-S18

48

Far $4-S15 815-819 $4-S19 S19-$20 S15-S18

% E/aceeptor 8 7

Close $4-$20 $4-S18

% E/aeceptor 31 26

S13-S19

25

$4-S16]17 815-S16/17

20 2O

S13-$20

20

Very far S14-S19 $4-S18

% E/acceptor 4 1

S13-S16]17

0

6

Underlined values are the most reliable since they are the average of the results of two or more samples which showed reasonable consistency. In eaaes like $4-$20 and S16]17-20 where lightly labeled $20 gave anomalous results, these values were not included in the average. given in Table 12. Lightly labeled samples which showed lower efficiencies t h a n more heavily labeled equivalent pairs are omitted. N o t all the results shown in Table 12 are equally reliable at the present time and this is so indicated. The striking feature of all of the d a t a is t h a t the observed efficiencies appear to fall into classes. Whether this is simply an accident of the particular pairs of proteins chosen for these experiments remains to be seen. B u t t a k e n at face value, the occurrence of such well defined classes argues t h a t the arrangement of ribosomal proteins m a y approximate some kind of regular array. To assign some meaning to the classes in Table 12, measured energy transfer has been used to compute distances between pairs of proteins. The procedures involved in such computations are described in Materials and Methods. Two different models have been used. One extreme is to assume random surface labeling of spherical proteins. Then the observed transfer efficiency will be a function of the radii of the two proteins, the distance between their centers, the mean number of acceptors per protein, and the characteristic transfer distance, Ro, for the pair of fluorescent dyes. One computes the transfer efficiency expected for given values of these variables and compares observed results graphically as shown in Figure 3. Most of our results are quite consistent with the expected dependence of efficiency on the number of acceptors. The results in Figure 3 show clearly the large range of distances covered b y our experimental data. The other extreme model is to assume t h a t all labeled proteins contain dyes located at a single, unique point. I n this case a graphical d a t a analysis is still carried out just like t h a t shown in Figure 3. Now, however, the results derived represent the distance between the locations of the eovalently attached dyes. This point to point measurem e n t can be fairly precise b u t is difficult to interpret in the absence of detailed structural information a b o u t ribosomal proteins. Distances calculated using b o t h models are given in Table 13. The numerical agreement between center to center and point to point results is excellent b u t this doe~ not resolve all ambiguities in the interpretation

464

K.-H. HUANG, R. H. F A I R C L O U G H AND C. R. CANTOR 0.8 0"7 ~

0.6

i~ 0.4

0-2x~ o x oQm

I I

I 2

......

F~G. 3. Analysis of typical energy transfer results to yield distance estimates. Observed efficiency ~, is plotted against the mean number of accepters per 30 S particle for the following pairs: S16/17-$20 ( × ), $4-813 (n), $4-$20 ({3), $4-s15 (A), $4-S19 (Zl). The first is in the very close category of Table 12; the next two in the close category, and the last two in the far category. :From the known molecular weights, pairs can be represented approximately by two spherical surfaces, one with a radius of 0.4 R0 and one with 0.3 Re. The solid lines in the :Figure represent the transfer efficiency expected as a function of the distance between the center of the spheres in units o f / t o. By interpolation each data point can be converted to the distances shown in Table 13. The general fit of the data to the functional form predicted by theory is evident from the :Figure. Re is 42.5 A; FAis the number of accepters per ribosome given in Table 9. From the semi-quantitative results in Table 13 one can given a rough interpretation to the classes of measured efficiencies shown in Table 12. Protein pairs called " v e r y close" are probably in direct contact. Computed surface to surface distances are close to zero. The "close" category represents proteins which m a y not be in contact yet are near enough to be nearest neighbors in the 30 S particle. There is room for a strand of R N A between two proteins in the close category but there is unlikely to be room for a globular protein. The "far" category consists of proteins that are unlikely to be nearest neighbors unless they have very extended structures. The " v e r y far" category shows transfer efficiencies too small to make an accurate quantitative estimate. 4. D i s c u s s i o n When the distances estimated for 30 S protein pairs are compared with other knoxvn facts or indications about protein proximity, there is a gratifying pattern of consistency. Two of the four proteins we compute as having surfaces with closest approach of 15 ~ or less .have been cross]inked: $7-$9 (Lutter et al., 1974) and $6-S18 (C. G. Kurland, personal communication). Two other proteins with distances of closest approach in the range of 20 to 30 A have also been crosslinked in the 30 S particle: $4-S13 (R. R. Traut, personal communication) and S13-S19 (Lutter et al., 1974; Sun et al., 1974). The in vitro assembly map of Held et al. (1974) is probably the aspect of ribosome organization most familiar to the general reader. I t is instructive to compare the

$20 $4 $4 $4 $4 S19 S19 S13 S16/17 $20 $4 $4 $4 $4 $4 84 $4 $4 S15 S15 $7 $7 S19 S15/S19 $20 $20 S15 $6 $4 S15 S16/17 S19 S19

FITC

15 13 25 15 27 15 12 6 24 15 7 8 3 18 32 8 16 3 16 8 10 30 7 4 14 8 29 10 2 4 0 3 2

(%)t

Transfer efficiency

1.02 1.16 1.06 1.15 1-08 1.05 1.23 1.32 `<0.70 0.80 1.66 1.66 1.60 1.32 1.05 1.67 1.42 1.56 1.24 1.30 0.76 0.69 1.36 1.64 1.05 1.37 0.76 0.91 >1.90 1.63 >1.70 1.60 >1.75

R/Re 43 49 45 49 46 45 52 56 <30 34 71 71 68 56 45 71 60 66 53 55 32 29 58 70 45 58 32 39 >81 69 >72 68 >74 --------

52

64

31

54

66

51

70

32

51

48

46

9 15 11 15 12 14 21 25 `<0 0 37 37 34 18 7 38 27 33 19 21 0 `<0 29 41 16 29 1 7 >47 40 >39 39 ;>43 --------

23

35

0

20

33

13

36

0

20

14

12

Distance calculation r a n d o m surface labeling~ Center to center Closest a p p r o a c h (A) average A (A) average 1.07 1.17 1.09 1.17 1.12 1.08 1.22 1.29 `<0.30 0-95 1.58 1-60 1.53 1.29 1.11 1.61 1.38 1.49 1-23 1.25 0.91 0.89 1.30 1.60 1.07 1.30 0-90 0.99 2.06 1.60 >1.8 1.53 1.68

45 50 46 50 48 46 52 54 -<15 40 67 68 65 55 43 68 59 63 52 53 39 38 55 68 45 55 38 42 88 68 >76 65 71

50

62

38

52

63

49

67

<27

51

49

47

D i s t a n c e calculation, specific labeling P o i n t to p o i n t R[Ro (A) average

t Measured value. T h i s also identifies t h e particular sample, described f u r t h e r in t h e legends to Tables 9 to 11. See t e x t for description. Center to center distance refers t o t h e relative location of t h e centers of t w o spherical proteins; closest a p p r o a c h refers to t h e p r o x i m i t y of two spherical surfaces. § Calculated using average emciency a n d average dye c o n t e n t for two samples s h o w n in earlier Tables.

$4 S2O $20 S13 S13 S13 S13 S19 $20 S16/17 S19 S19 S19 S16]17 S16/17 S15 S15 .S15 S16/17 S16/17 $9 $9 S15 §S15/S19 S13 §S13 $8 $18 S18 S18 S13 $20 S14

Sample I-AEDAIqS

Distance estimates between Tairs of 30 S ribosomal proteins

'I'ABL~E 1 6

466

K.-H.

HUANG,

R . H. F A I R C L O U G H

A N D C. R . C A N T O R

(c)

S21

(b) FIG. 4. (a) Schematic summary of all of the average distances between the ribosomal proteins shown in Table 13. Protein radii and distances are drawn roughly to scale. Thick solid lines indicate distances, t h i n lines are lower limits. To display the d a t a in two dimensions, distances between S15-S19 and S19-$20 h a d to be increased,artificially. This is shown b y the dashed lines. ( b ) / n vitro assembly map of the 30 S particle, adapted from Held e~ aL (1974). Circled proteins are the subset for which most energy transfer d a t a is currently available.

ENERGY TRANSFER IN RIBOSOMAL PROTEINS

467

pattern of proximities we have observed with the assembly map. This is done in Figure 4. Distances in this Figure are the average of the results reported in Table 13 as analyzed by the surface to surface model. Consider first $4 protein. I t is linked in reassembly to S13, S16/17 and $20, but not to S15, S18 or S19. Our distance estimates indicate that $4 is much closer in space to each of the three former proteins than to the three latter. This general pattern of agreement is repeated by most of the measurements shown in Figure 4. A notable exception is S13-S19. These proteins are not ]inked in the assembly map of Held et aL (1974). However, we find them close and others cited above have been able to cross]ink them. S19-S14 and S15-S18 are ]inked in assembly, but energy transfer studies suggest they may not be located near each other. While $8 and S15 are not linked in the assembly map, partial nuclease digestion studies have indicated they may be near neighbors (Morgan & Brimacombe, 1973) and indeed we find them very close. An interesting fact emerges when the results in Figure 4 are examined more closely. Many protein pairs, such as S15-S18 and S13-$20, linked in assembly are not close enough to make extensive protein-protein contact very likely. Therefore, assembly linkage implies proximity but not necessarily contact. Presumably, the assembly linkages are mediated in many cases through effects of the intervening rRNA. Sufficient energy transfer data exists ah'eady on a subset of six of the 30 S proteins to allow some tentative conclusions about their arrangement in space. For the proteins $4, S13, S15, S16/17, S19, $20, twelve distance estimates are available (see Fig. 4 and Table 13). If the proteins were spherical, 14 distances would be required to specify the structure out of 15 possible total protein pairs. We have only 12 distance measurements and four possible structures emerge which are consistent with all of our available data. These correspond to various puckered conformations of the pattern shown in Figure 4(a). A significant feature of all four models is their deviation from planarity. If the proteins are spheres, calculated minimum thicknesses range from 68 A to 79 A. This argues that the simple and attractive hypothesis that the 30 S particle is a planar lattice of proteins (Hill & Fessenden, 1974) is ~mlikely. A second feature of all models is the amount of space not occupied by the proteins. Some of this may be the site of other proteins but it is highly tempting to speculate that much of it is the location of portions of the 16 S rRNA. This would agree well with earlier suggestions made by Kurland (1974) and the known ability of proteins fike $4 to interact with vast regions of the 16 S rRNA (Zimmerman et al., 1972; Schaup e~ aL, 1971; Schaup & Kurland, 1972; Nanninga et aL, 1972). All of the above discussion is based on data analyzed by the sphere to sphere model. Suppose instead that all proteins had been specifically labeled at unique points. Since the distance results are almost identical, the schematic data summary of Figure 4 would still apply. However, now all that one can say is that there is a portion of each protein located at the center of the spheres shown in that Figure. Note that this is unlikely to change the qualitative conclusions about non-planarity and the degree of open space. The details of any quantitative model that might be constructed would, of course, be altered considerably. A more serious difllculty arises if the proteins, instead of being globular, have very extended structures. Indeed two very recent studies indicate this may be the case for at least some 30 S proteins both in solution (Rohde et aL, 1975) and in the 30 S particle (Lake et aL, 1974). I f this turns out to be a general feature, interrelations of distances based on the sphere to sphere model can have no validity. Instead it will be necessary

468

K.-H. HUANG, R. H. F A I R C L O U G H AND C. R. CANTOR

to determine the actual location of covalently attached dyes within the plimary structure of each protein. Then discrete point to point models will yield specific distances. Naturally this will involve considerably more effort and furthermore m a n y such measurements would be needed to establish the relative spatial orientation of each protein pair. However, such experiments, though dif~cult, should be feasible as the results presented here have demonstrated. An example of how these could proceed is given b y the results for I - A E D A N S $20 to F I T C - S 4 and I-AEDANS-S20 to FITC-S16/17 energy transfer reported in Table 10. Recall t h a t these showed high efficiency with heavily labeled $20 and low or no efficiency with lightly labeled $20. This must be an indication t h a t the I - A E D A N 8 reaction with 820 is not random. We can estimate the minimum length for $20 in the 30 S ribosome as follows. Suppose t h a t the first I - A E D A N S to react with 820 goes on to a fixed point as far away from $4 and 816/17 as possible. Suppose t h a t subsequent I - A E D A N S reaction takes place at a different fixed point as close to 84 and S16/17 as possible. Since the distance between 84 and S16/17 is known, the transfer efllciencies between each of these two and both the lightly labeled and heavily labeled 820 can be used to calculate the positions of the two sites of labeling in 820 (see ~/g. 5). This leads to a minimum distance between these two positions of 45 A. I f 820 were spheri-

I

......~'~

S 20

S 16/17

S4

I~IG. 5. Estimation of the minimum length of protein $20. The three circles are in the positions of proteins $4, S16/17, and $20 determined from distance measurements assuming random spherical distributions of dyes. These oorrespond to the same structure shown in Fig. 4. Proteins are drawn to scale. If instead one assumes 2 unique labeling sites on $20, distances to each of these sites are calculated from the data in Table 10 using the point to sphere model of Ge,~ni~ & Cantor (unpublished results). The results are: first reactive $20 site (---) > 96 • to the centre of $4; 54 A to the center of S16/17; subsequent reactive site (...) 51 ~- to $4; 19 A. to the center of S16/17. This leads to an elongated $20 shown as an ellipsoid bounded by the two sites.

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469

cal, the diameter would be a b o u t 30 A. So our results suggest t h a t $20 is likely to have an1 axial ratio of at least 2:1 in the 30 S particle. I n conclusion, the studies described here h a v e demonstrated the usefulness of energy transfer measurements to determine the spatial arrangement of ribosomal proteins. Compared with crosslinking, the principle advantage of energy transfer is t h a t distant protein pairs can be found in addition to proximate ones. Furthermore, the semi-quantitative distance measurements t h a t result are essential when an actual structure rather t h a n just a schematic one is to be constructed. However, interpretation of both energy transfer and crosslinking measurements will be made very complicated b y a n y pecularities in the spatial distribution of reactive side chains of the 30 S proteins. The authors are very grateful to 1~I. Nomura and W. Held for providing extremely valuable advice on protein purification. They also kindly donated protein samples and collaborated in early studies which showed the feasibility of reassembling dansyl a~d fluorescein-labeled ribosomal proteins. The excellent technical assistance of Elaine Lin is gratefully acknowledged. This work was supported by grants from the U.S. Public Health Services (GM19843) and the National Science Foundation (GB 34522X). REFERENCES Beardsley, K. & Cantor, C. R. (1970). Proc. Nat. Acad. So/., U.S.A. 65, 39-46~ Bickle, T. A., Hershey, J. W. B. & Traut, R. R. (1972). Proe. Nat. Acad. Sci., U.S.A. 69, 1327-1331. Bunting, J. R. & Cathou, R. E. (1973). J. Mol. Biol. 77, 223-235. Chang, F. N. &Flaks, J. G. (1972). J . Mol. Biol. 68, 177-180. Craven, G. R., Voynow, P., Hardy, S. J. S. & Kurland, C. G. (1969). Biochemistry, 8, 2906-2915. Dale, R. E. & Eisinger, J. (1974). Biopolymers, 13, 1573-1605. Edman, P. (1956). Acta Chem. S c a ~ . 10, 761-768. FSrster, T. (1965). I n Modern Quantum Chem. Leer. Istanbul Int. Summer Seh., 1964 (Sinanoglu, 0., ed.), pp. 93-137. Garrett, R. A. & Wittman~, H.-G. (1973). Endeavour, 32, 8-14. Gennis, R. B. & Cantor, C. R. (1972). Biochemistry, 11, 2509-2517. Gennis, L. S., Gennis, R. B. & Cantor, C. R. (1972). Biochemistry, 11, 2517-2524. Green, M. & Kurland, C. E. (1971). iVa~ure N e w Biol. 234, 273-275. Held, W. A., Ballou, B., Mizushima, S. & Nomura, M. (1974). J . Biol. Chem. 249, 3103-3111. Hill, W. E. & Fessenden, W. F. (1974). J . Mot. Biol. 90, 719-726. Hill, W. E., Rossetti, G. P. & Van Holde, K. E. (1969). J . Mol. Biol. 44, 263-277. Hosokawa, K., Fujimura, R. K. & Nomura, M. {1966). Proc. Nat. Acad. Sci., U.S.A. 55, 198-204. Hsiung, N. & Cantor, C. R. (1973). Arch. Biochem. Biophys. 157, 125-132. Huang, K. & Cantor, C. R. (1972). J. Mot. Biol. 67, 265-275. Huang, K. & Cantor, C. R. (1975). J. Mol. Biol. 97, 423-441. Hudson, E. (1970). Ph.D. dissertation, University of Illinois. Kaltschmidt, E. (1971). Anal. Biochem. 43, 25-31. Kinard, F. E. (1957). Rev. Sei. Instr. 28, 293-294. Kin'land, C. G. (1972). A n n u . Rev. Biochem. 41, 377-408. Kin'land, C. G. (1974). I n Ribosomes (Nomura, M., Tissi&res, A., & Lengyel P., eds), pp. 309-331, Cold Spring Harbor Press, New York. Lake, J. A., Pendergast, M., Kahan, L. & Nomura, M. (1974). Proc. Nat. Acad., U . S . A . 71, 4688-4692. Lowry, O. H., Rosebrough, N. J., Farr, L. & Randall, R. J. (1951). J . Biol. Chem. 193, 265-275. Lutter, L. C., Zeichhardt, H., Kurland, C. G. & StSffier, G. (1972). Mot. Gen. Genet. 119, 357-366.

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