Tertiary structure of bacteriorhodopsin

J. Mol. Biol. (1989) 210, 829-847

Tertiary Structure of Bacteriorhodopsin Positions and Orientations of Helices A and B in the Structural Determined by Neutron Diffraction

Map

Jean-Luc Popott Institut de Bioloqie Physico-Chimique and Coll&qe de France, 13 rue Pierre et Marie Curie F-75005 Paris, France

Donald M. Engelman Yale University,

Department of Molecular Biophysics New Haven, CT 06511, U.S.A.

& Biochemistry

Ogan Gurelt and Giuseppe Zaccai’ Institut

(Received

Laue-Lange&n 156X, F-38042

4 April

and CNRS U.A. Grenoble, France

1333

1989, and in revised form 10 August

1989)

Positions and rotations of two helices in the tertiary structure of bacteriorhodopsin have been studied by neutron diffraction using reconstituted, hybrid purple membrane samples. Purple membrane was biosynthetically 2H-labeled at non-exchangeable hydrogen positions of leucine and tryptophan residues. Two chymotryptic fragments were purified, encompassing either the first two or the last five of the seven putative transmembrane segments identified in the amino acid sequence of bacteriorhodopsin. The 2H-labeled fragments, diluted to variable extents with the identical, unlabeled fragment, were mixed with their unlabeled counterpart; bacteriorhodopsin was then renatured and reconstituted. The crystalline purple membrane samples thus obtained contained hybrid bacteriorhodopsin molecules in which certain transmembrane segments had been selectively 2H-labeled to various degrees. Neutron diffraction powder patterns were recorded and analyzed both by calculating difference Fourier maps and by model building. The two analyses yielded consistent results. The first and second transmembrane segments in the sequence correspond to helices 1 and 7 of the three-dimensional structure, respectively. Rotational orientations of these two helices were identified using best fits to the observed diffraction intensities. The data also put restrictions on the position of the third transmembrane segment. These observations are discussedin the context of folding models for bacteriorhodopsin, the environment of the retinal Schiff base, and site-directed mutagenesis experiments.

1. Introduction

7 Author to whom all correspondence should be sent. $ Present address:ColumbiaUniversity, Collegeof

It is now more than a decade since the lowresolution structure of bacteriorhodopsin (BRQ;) revealed that it has seven transmembrane helices (Unwin & Henderson, 1975; Henderson & Unwin, 1975), and almost as long since the amino acid sequence was determined (Ovchinnikov et al., 1979; Khorana et al., 1979). Seven putative trans-

Physicians & Surgeons, New York, NY 10032, U.S.A. Q Abbreviations used: BR, bacteriorhodopsin; C-l, chymotryptic fragment of BR, residues 72 to 248; C-2, chymotryptic fragment of BR, residues I to 71; D,-PITC, ‘H-labeled phenylisothiocyanate; e.m., electron microscopy; n.m.r., nuclear magnetic resonance; PM, purple membrane; s.D., standard deviation. 0022-2836/80/240829-19

$03.00/O

829

0 1989 Academic Press Limited

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J.-L.

Popot et al.

STAAnBoB

!+a

PEP6E,,

sH R P E U Ab

.. op

E G

(b) Figure 1. (a) Transmembrane folding model of BR (from Engelman et al., 1982). Residues that carried ‘H in the present experiments are indicated by filled squares. Chymotrypsin cleaves,~bacterioopsin after Phe71 (\). Fragment C-Z covers residues 1 to 71, fragment C-l covers residues 72 to 248. (b) Distribution of ‘H atoms in the 7 helices, projected on a plane normal to the axis of the helix (shown by + ).

membrane segments have been fairly narrow limits of uncertainty

identified within by hydrophobi-

city analysis of the sequenceand a number of modification or immunolabeling studies (Engelman et al., 1982,

1986;

and

references

therein).

The

aim

of

obtaining a chemical model for BR by combining the structural and sequence data has been approached by a variety of means, yet no reliable positioning of the polypeptide chain in the electron density map has emerged so far. Neutron diffraction coupled with LH labeling has been used to identify features of the molecule in planar projection, most notably the position of the retinal chromophore (Jubb et al., 1984; Seiff et al., 1985, 1986~~; Heyn et al., 1988). As the retinal is

linked by a Schiff base to lysine 216, which lies in helix 7, these studies place constraints on the possible location of that part of the polypeptide chain. Further modification or labeling studies have been directed to other parts of the molecule (see Discussion). To this day, however, attempts to position any of the transmembrane sequence segments have been plagued by major uncertainties. BR has been labeled biosynthetically by incorporation of ‘H-labeled amino acids. Analysis of the neutron diffraction patterns followed two strategies: difference Fourier methods (Engelman & Zacca;i, 1980) and model building (Trewhella et al., 1983). Each approach involved complications arising from intrinsic

methodological

limitations

and

from

the

Path of the Polypeptide

831

Chain in Bacteriorhodopsin

Table 1 Flow-chart

of experiments

(1) Purijicution

of BR fragments

pz,

g

$zjfzji-i

(2) Reconstitution (sample

of hybrid number)

Unlabeled C-l Unlabeled C-2 Per-‘H-labeled C-2 C-2 labeled on Leu and Trp C-2 labeled on Leu and Trp+ unlabeled C-2

samples

C-l labeled on Leu and Trp

C-l labeled on Leu and Trp + unlabeled C- 1

655

660, 661

267, 657 272 654 658, 659 (3) Recording

of neutron

diffraction

patterns

(4) Analysis

Comparison

of experimental

fact that’ the label was spread throughout the molecule. A further difficulty with the earlier modelbuilding approach was the large number of models that needed to be considered (greater than 10’). The analysis required a stepwise screening that risked eliminating the correct folding model at an early stage. In the present work, we have reduced the combinatorial problem and restricted the label distribution by reassembling BR molecules from two fragments, one of them ‘H-labeled and the other not. A flow-chart of the experiments is shown in Table 1. We have shown previously that two-dimensional crystals of BR with the native structure and geometry can be reformed following renaturation of BR from these fragments (Popot et al., 1986, 1987). Fragment C-2 contains the first two of the transmembrane helices and fragment C-l the last five (Fig. 1). ‘H can be introduced selectively into one of the two fragments. As an initial effort in this direction, we per-2H-labeled the two-helix fragment (C-2) and localized it to one end of BR structural map (Popot et al., 1986; Trewhella et al., 1986). It was not possible, however, to determine a unique position for each of the two helices nor to define their rotational orientation. The improvements included in the present work concern sample preparation and data analysis. By labeling only the non-exchangeable positions of

and predicted

maps

leucine residues and the u and /3 carbon atoms of tryptophan residues, the label was confined to the transmembrane region. Moreover, helices that were not labeled to the same extent could be distinguished one from another. Hybrid BR samples were constructed with either the C-l or the C-2 fragment 2H-labeled to various extents. Because of the weaker labeling, data analysis could be carried out by both difference Fourier methods and model building. The model-building methodology developed for the present work followed the strategy used by Jubb et al. (1984) for localizing 2H-labeled retinal; model data are not calculated entirely ab initio, but by combining structure factors derived from intensities observed with native purple membrane (PM) and structure factors calculated for the label. This approach has two advantages over that used by Trewhella et aE. (1983, 1986); it minimizes errors due to approximations in the model and it reduces considerably the computational cost of extensive model searches. The present work establishes the positions of each of the first two transmembrane sequence segments in the structural map and provides some indication of the probable position of the third segment. It defines a preferred rotational orientation for these helices about their axes. An overall folding model in which transmembrane helices pack without interleaving, as originally suggested by Engelman et al.

832

J.-L.

Popot

is consistent with (but not uniquely established by) the new data and many of the other studies in the literature. The present results give indications as to which amino acid residues are likely to contribute to the environment of the retinal Schiff base, a region that is thought to play a key role in the mechanism by which BR uses light energy to transport protons. (1980),

2. Materials

and Methods

(a) Sample preparation

and analysis

BR fragments were prepared following the protocol of Liao et al. (1983) as modified by Popot et al. (1987), starting from unlabeled PM and from PM purified from cultures grown on a defmed medium (Crespi, 1982) in which leucine and tryptophan were substituted with their fully ‘H-labeled counterparts (MSD Isotopes, St Louis, MO). L-[3,4,5-3H(N)]leucine (New England Nuclear) was added in trace amounts to the main cultures and at a spec. act. of 750 cts/min per nmol leucine to a smaller fraction of the ‘H-labeled culture. The radioactive spillover to other amino acids was determined by running a hydrolyzed sample of PM from the small culture on a Beckman 121M amino acid analyzer and fractionating the effluent prior to its entry into the reaction coil. No contamination was found above the threshold of detection ( ~01% of the radioactivity in leucine), with the possible exception of lysine (01%). Altogether, the radioactivity in amino acids other than leucine represented less than 1.7% of that in leucine. The level of 2H-labeling in the final preparation of fragments could not be determined precisely. Preliminary ‘H and ‘H n.m.r. data indicated that the level of incorporation was very high, except for the aromatic 2H atoms of the tryptophan residue, which had been totally exchanged for ‘H during solubilization of the fragments in formic acid (Bak et al., 1967). The level of leucine incorporation estimated from the specific activity of leucine in the PM from the small growth (approx. 40%) was lower than expected from the n.m.r. data and from earlier determinations on similar cultures (77%; Trewhella et al., 1983). The cause of this discrepancy was not resolved, and all model calculations had to be repeated using a wide range of 2H-labeling levels. The level of incorporation of ‘H into tryptophan was assumed to be similar to that in leucine, as suggested by the n.m.r. data. Large errors on this level would have little influence on the calculations, since tryptophan residues contained only 18 ‘H-labeled positions out of 268 in the C-l fragment, and 6 out of 116 in the C-2 fragment. The fragments were analyzed by SDS/polyacrylamide gel electrophoresis as described (Popot et al., 1987). They all contained less than 2% of the other fragment or intact BR. Seven new large-scale samples of hybrid PM were prepared, as summarized in Table 2. In some caes (samples 658 to 661), the ‘H-labeled fragment was diluted prior to renaturation with the identical, unlabeled fragment, to the molar ratio indicated. All samples were prepared by the standard method of Popot et al. (1987), using 1 : 1: 1 (by weight) protein/HaZo&terium lipids/ taurocholate proportions and a 1 : O-9 : 1.25 C-l/C-2/retinal molar ratio. They were spread over 1 or 2 quartz slides and partially dehydrated over a saturated solution of CaCl, at room temperature as described (Popot et al., 1986). The mosaic spreads of the samples were between 10” and 20” (full-width at half-height).

et al. (b) Data collection The sample quartz slides were mounted vertically in an aluminum can on the sample axis of the D16 diffractometer at the Institut Laue Langevin in Grenoble. The temperature was regulated at 20°C and the relative humidity was maintained at 32% by enclosing in the oan a bath of saturated CaCl, solution. These conditions had been established previously to be best for data collection from samples of reconstituted bacteriorhodopsin (Popot et al., 1986, 1987). The diffractometer experimental design and the treatment of observed intensities were similar to those described by Jubb et al. (1984). Neutrons of wavelength 452 A (1 A=01 nm) from a graphite monochromator passed through a beryllium filter cooled by liquid nitrogen and were collimated by 2 sets of cadmium slits @7 m apart (45 mm x30 mm close to the monochromator and 3 mm x 20 mm close to the sample). Data were collected by modified 8/26 scans, the setting of o, t,he angle between the direction of the beam and the normal to the quartz plates, always being equal to half the value of y, the angle between the incident beam and the line joining the sample to the center of the multidetector. The multidetector covered an angle of about 10” in 2% with a wire resolution of @146”. It was scanned in steps of 06” and the data correspcnding to the same 28 value were averaged. This approach made full use of the possibility of simultaneous recording over an angular range in 26. and compensated for fluctuations in counting efficiency across the detector face. Observed intensities were multiplied by a Lorentz factor equal to (h2 + hk+k2)* after subtraction of a background value estimated from the values on either side of each peak. Experimental errors were assessed as described below. The correction due to the differences in absorption and amount of material in the beam between the 2 extremes of the scan was negligible.

(c) Model-building

analysis

(i) Model building Model building followed the general approach developed by Jubb et al. (1984) for locating ‘H-labeled retinal. Structure factors predicted for a given folding model are calculated by vectorial addition of structure factors for the native PM and structure factors calculated for the label. Structure factors for the native PM were estimated from the peak intensities (integrated areas) of the neutron diffraction pattern and from the phases determined by analysis of e.m. images (Henderson et al., 1986), according to the approach of Zaccai’ & Gilmore (1979). When reflections overlap, they were split according to the electron diffraction intensity ratio. The treatment of errors introduced by these assumptions is described below (subsection (cl (ii) PI). Structure factors for the label were calculated by taking into account only those ‘H nuclei known to be substituted with 2H. Transmembrane segments were delimited as indicated in Fig. l(a) (see also Engelman et al., 1982, 1986). Alternatively, helix A could be moved up or down the sequence by 1 residue, helix B could be moved up by 1 residue or down by up to 5 residues (thus including Leu66 in the model) and helix F could be moved up by 6 residues. Hence, Leu201, in the loop between helices F and G, is the only labeled residue that w&8 never included in the calculations. The position of the 2H atoms in each assignment model and the resulting partial structure factors were calculated as described by Trewhella et al.

Path of the Polypeptide

833

Chain in Bacteriorhodopsin

(1983) for complete BR models. Structure factors for native PM and for the label were then added after appropriate scaling (see below) and the predicted reflection intensities calculated and, in the case of overlapping reflections, summed. There are both theoretical and computational advantages to this new approach when compared to that used previously (Trewhella et al., 1983, 1986): (1) Only the contribution of the label is calculated ab in&o, so that errors in calculating the contribution of the unlabeled atoms in the transmembrane helices are not introduced in the predictions. Furthermore, the contribution of unmodeled (extramembrane) parts of the molecule is contained in the experimental native structure factors, while our label was essentially restricted to the transmembrane segments (106 to 116 ‘H atoms out of a total of 116 when C-2 was labeled (depending on the limits assigned to helix B), 258 out of 268 when C-l was labeled; see Fig. l(a)). Therefore, the predicted intensities represent, in principle, the entire PM with its modifications, and not just the transmembrane domain of BR as in the earlier approach. (2) From a practical point of view, the new procedure requires that for each model the contribution of only 2 helices (samples 654) or 5 helices (samples 655 and 660) be considered. Permutations and rotations of unlabeled helices do not alter predicted intensities, as they did with the previous approach. This diminishes enormously the number of models to be tested; complete searches, involving the 5040 assignment models and 360” rotations by 60” steps of all helices, previously required testing over 1.9 x lo9 models. The new procedure divides this number by factors of 9 x 10’ (2 labeled helices) and 72 (5 labeled helices). The predicted intensities for the native molecule, being the experimental values, do not have to be calculated for each model, which again cuts down on computing time by a factor close to 2. Combined with the use of the faster computers of the CIRCE computing center, these savings on computing time rendered a fine screening of all possible models practical, as well as systematic explorations of many potential sources of error. (ii) Assessment of errors There are two kinds of error to be taken into account, errors in the measurements and errors inherent in the model calculations. (ii)(a) Experimental errors Standard deviations based on counting statistics underestimate experimental uncertainties on several grounds. They do not take into account possible variations from sample to sample, nor errors arising from the determination of peak areas. These can be introduced while estimating local background and partial overlaps with other peaks. In order to minimize variations due to human errors, all of the peak areas used for the present work were measured by the same investigator. Rigorous determination of overall experimental errors on peak intensities would call for several full sets of data to be collected for each sample, which would require a prohibitive length of beam time. Estimates were obtained by comparing I4 partial sets of data, recorded on 6 control samples and 3 labeled samples. Differences among unlabeled reconstituted samples were no greater than between unlabeled reconstituted samples and native PM, and were similar to differences between independent sets of data recorded from the same sample. Reflection intensities for 7 sets of control data were first normalized

to the same sum. The control samples used for this determination included: native PM; 2 samples (267 (2 independent data sets) and 657) reconstituted from native fragments; and 3 samples (658,659 and 661) reconstituted using a mixture of unlabeled and 2H-labeled fragments (see Table 2). The amount of label in the latter 3 samples turned out to be too low to change detectably the diffraction pattern, differences from control samples remaining within the range of those observed among control samples. These -data were used as further control data. This may have contributed to a slight overestimate of the errors. Six pairwise comparisons were made by calculating ratios (r) of normalized intensities (the 2 sets of data on sample 267 against each other, and each remaining set against our reference sample, i.e. the sum of the 2 measurements on 267). Reflections that could not be measured precisely enough in a given partial set were excluded from the comparison. The relative standard error on each reflection was taken to be equal to (l/n,/Z)-’ Z]r- I], w h ere n is the number of comparisons. Altogether, the procedure is likely to yield a somewhat if only because intensity measureoverestimated S.D., ments on partial sets are less accurate than on complete sets. Fig. 2 shows a comparison of the data collected on sample 267 with data from PM. The relative S.D. on the ratio of intensities has been taken to be 42 times the relative S.D. on each intensity. The fact that all ratios but one fall within 1 S.D. of r= 1 confirms that our estimate of the accuracy of the data is conservative. This is reflected in the value of the reduced x2 obtained when comparing native PM and sample 267, xt =@69, which indicates that the agreement between the 2 sets of data is slightly better than statistically expected for 2 samples taken from the same population (x,” = 1). Differences between independent, partial sets of measurements on labeled samples were very close to those expected from the averaged S.D. values. Exceptions were reflections, particularly 3,0, that are much weaker in control wersu8 labeled samples. In such cases, relative errors were, as expected, smaller than for the controls. As a rigorous estimate was difficult, relative errors on these reflections were nevertheless taken to be the same as for control samples. As a result, their weight in the model calculations was slightly diminished. (ii)(b) Errors in calculating predicted intensities Predicted peak intensities I, for labeled samples were calculated as: 1, = C [Fn,j12 = C [F,,j+(F,,jx j j

SCALE)]‘,

(1) where Fn , j is the structure factor for reflection j, F, , j is that part of the structure factor due to the native structure, F,,j that calculated for the label, SCALE is a scaling factor, and the sum is taken over the j reflections that overlap in the powder pattern, when applicable. Errors in this calculation arise from the following sources: (1) errors in determining F,, which are due to errors on the measurement of peak intensities for unlabeled PM, errors due to splitting overlapping reflections according to electron diffraction measurements, and errors due to using e.m. phases; (2) errors in calculating F,, and (3) error in scaling the contribution of the label to that of PM. Errors in the measured intensities used to calculate F, were estimated as described above (subsection (c)(ii)(a)). They were taken into account in the evaluation of the models by affecting predicted and observed reflection intensities with the same relative error (see subsection (iii) below).

834

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Popot et al.

The effect of errors due to using e.m. phases is difficult to treat analytically (Jauch, 1987). At a resolution of 7 A, the uncertainty of experimental e.m. phases is only a few degrees (Henderson et al., 1986) and was neglected. It has been pointed out that, at the resolution of the experiments, the contrast between protein and lipids in the structure is similar for neutrons and for electrons, which should result in phases being similar (Zaccai’ $ Gilmore, 1979). In a comparison of calculated electron and neutron diffraction phases for a dozen models taken at random, we found that the unweighted average phase difference was only 13” over the range from reflection 1,l to reflection 7,1, with most of the large deviations affecting the weakest reflections. This result is consistent with that of a similar calculation using a simplified 2-dimensional model (PlGhn & Biildt, 1986). In order to estimate the effect of these deviations on our analysis, we repeated critical searches after altering the e.m. phases by adding errors of the same magnitude as the average deviation between neutron and electron diffraction phases, with random signs. The effect of errors due to using the e.m. ratios for the separation of overlapped reflections was tested in a similar manner. Jauch (1987) has calculated that, in the absence of any information, Fourier maps of an acentric structure involving pairs or triads of overlapped reflections would have the minimum error if the reflections in a group were set to be equal, the average error being then 24 to 29% of the total intensity. Errors resulting from using the e.m. ratios are bound to be lower, as the electron and neutron diffraction patterns are similar. For an average of a dozen models, we compared neutron diffraction intensities calcuiated by summing the intensities for overlapped reflections and splitting them according to the calculated e.m. ratio and neutron diffraction intensities calculated directly for each reflection. The unweighted average relative difference over the range from reflection 1,l to reflection 7,l was 20%. As a check that this source of error did not bias our conclusions, we repeated calculations after affecting observed intensities for overlapped reflections with an extra relative error that was varied between 0 and 40% (see Results). Errors in calculating the structure factors for the label, due to inadequacies in the model, are difficult to estimate. Trewhella et al. (1983) reported that conventional crystallographic residuals (Zl#‘,,i - F,,ljXF,,) between observed (P,) and calculated (P,) electron diffraction structure factor moduli were in the range 29 to 34%, indicating that the model is only moderately successful in predicting native structure factors. It should be remembered that the model takes into consideration only the transmembrane part of BR, to the exclusion of the connecting loops and retinal. Transmembrane segments are ideal, straight q-helices, with side-chains arbitrarily set in extended conformations. In the present work, the model is being used only to calculate the contribution of selected amino acid residues in the transmembrane region, so that it must be expected to fare better than the above data would suggest. Preliminary calculations indicated that setting the side-chains in more realistic conformations slightly improved the predictions (R. Lavery t J.-L.P., unpublished results). Nevertheless, imperfections in the model probably represent one of the most serious limitations of the model-building approach. In most computations, helix rotations were varied by 60’ steps. In some cases, 120” steps were used. The effect of such a coarser screening is to increase randomly the 1: for the best rotational variant of each assignment model. For a xz distribution like that shown in Fig. 3(a), the

expected increase over the best x,’ observed in a search by 60’ stops would generally lie between 0 and 1, with occasional increases by up to 2. While differences between assignment models would become blurred, the best ~3 for the AlB7 assignment model would still be within, at most, 2, and most likely within less than 1, of that for the assignment model featuring the best overall ~5. This effect was indeed observed when comparing the results of 6 searches on the data from sample 654, starting from different initial rotational positions and examining all assignment models; on average, the best x,’ for each assignment model increased by 945( +055) when helices A and B were rotated by stops of 120“ instead of 60”. The greatest difference between the best assignment model and model AlB7 increased from 94 to 69. Finally, errors in scaling the native, observed structure factors to those calculated for the label arise from 2 sources. First, due to material difficulties, the extent of ‘H-labeling of the peptides could not be determined precisely (see above). Second, the contribution of unmodeled regions to the overall diffracted intensity in the native powder pattern is unknown (although probably small at the present 7 A resolution, as connecting loops are not revealed in the 3-dimensional Fourier synthesis). The native structure factors F, (calculated from observed intensities) were scaled so that the sum of the observed intensities would be identical with the average sum predicted for a number of randomly taken. unlabeled models calculated ab in&. Assuming 100c/o ‘H-labeling and no contribution of the non-modeled regions would imply a SCALE factor equal to 1 in eqn (1). However, a value for SCALE of 66 to 68 is probably more realistic and was used in most calculations. As indicated in Results, none of our conclusions was sensitive to the value of SCALE (varying over a range of 91 to 1). (iii) Assessment of folding models The ability of folding models to predict observed intensities was assessed by x2 analysis. gnother parameter, R, had been described (Trewhella et al., 1983). This parameter R had 2 drawbacks; namely, the manner in which the various reflections were weighted and the difficulty in estimating its statistical significance. In this earlier approach, errors on the data were taken into account by summing the statistical error resulting from counting statistics (which, for most reflections, is much smaller than actual experimental errors) with a constant, the same for all reflections, representing an estimate of systematic errors. As a result, R was dominated by the largest of them. For samples such as those here, where efficiently among models that correctly predict the 3 or 4 largest of them. For samples like those here, where labeling is low, most intensity variations are small, discrimination is more difficult, and the number of such models is bound to be larger than with the heavily ‘H-labeled sample that we used previously (Trewhella et al., 1986). In the present study, the usual statistieal parameter x1 was calculated so that the contribution of each reflection to the selection of models may be precisely weighted it9 a function of its accuracy:

(2) where ICI i and IO ,! are the calculated and observed intensities for reflection i, respectively, and (T, , i and a,, i are the associated standard deviations. 6, i (a,, i) was set equal to 1,. i (I,. i) multiplied by the relative S.D. on the

Path of the Polypeptide intensity of reflection i, estimated as described above and given in Table 3. I, is the intensity measured for native samples, which is used in the calculation of F, (see eqn (1)). The choice of these values for o,, i and u,, i is based on the facts that: (1) experimental errors were found to be the same for labeled or unlabeled samples (see above); (2) as F, is usually larger than F,, much of the error on I, comes from the experimental error on F,. The impact of the extra errors added when calculating and scaling F, and F, was examined ay described above. v was taken to be equal to N, the number of reflections taken into account for the calculation. For most calculations, N was about 20. The confidence interval within which the probability that a given model can account for the observations exceeds 5% extends to xz x 1.6, the 1 o/o confidence interval to x,’ % 1.9 (Bevington, 1969). In the examination of sample 654, where many reflections are close to native (control) values, it was observed that smaller values of x,’ were obtained when the SCALE factor (used to scale calculated F, to experimental F,; see eqn (1)) was lowered to unrealistic values (e.g. @15 to @30). This simply reflects the fact that lowering SCALE diminishes the contribution of the label to the calculated intensity I,, and raises the probability that I, will be close to I, for those reflections that show little or no variation. Typically, the xt value for a given model would decrease, e.g. from 1 to @7. This computational bias did not change the rank ordering of the best models, but introduced some uncertainty in the absolute values of xe. All calculations were performed both at the more realistic values of SCALE=@6 to @8 and at those giving the lowest 1: (015 to @3). None of our conclusions depended on the choice of SCALE. For the more heavily labeled sample 655, where most reflection intensities differ significantly from native intensities, this effect was not observed, minimal xz values being observed in the range SCALE = @6 to @7. The performance of the new model-building and statistical analysis as compared with that used previously (Trewhella et al., 1983, 1986) was tested by analyzing again the data collected on sample 272, in which fragment C-2 had been per-‘H-labeled (Popot et aE., 1986; Trewhella et al., 1986). The new analysis confirmed the previous conclusions regarding the assignment of 1 of the 2 labeled helices to position 7. In addition, models 1+7 and 6+7 were clearly distinguished from oneanother, which in the previous analysis (Trewhella et al., 1986) had yielded comparable values of R (cf. Results, subsection (b)(ii)(c)). (d) Fourier maps Fourier projection maps of the native structure were calculated from neutron diffraction factor moduli measured from native PM or from PM reconstituted with non-*H-labeled BR fragments (the intensities of the 2 samples being the same within errors) with phases taken from electron microscopy (Henderson et aE., 1986). Intensities corresponding to the sum of non-equivalent reflections (e.g. the 2,l and the 1,2) were split according to the ratio of intensities in the electron diffraction pattern (Henderson et aE., 1986). For observed difference Fourier maps, data sets were first scaled to the same sum of intensities over the 1,l to 5,2 range. Because the ‘H content of samples varied, a further scale factor was included, which was estimated from structure factor calculations on model structures having the appropriate ‘H levels. As this scale factor was somewhat model-dependent, it was always verified that the main features of the maps did not vary when the scale factor was changed within the limits obtained for the different models with the same ‘H levels. The scale factor

Chain

835

in Bacteriorhodopsin

was further checked by verifying that density fluctuations in the lipid regions of the unit cell were minimal, as expected since differences in ‘H-labeling were on the protein only. Two approaches were used to calculate the structure factor amplitudes of overlapped powder reflections for the- difference Fourier maps: in the lst, the intensity difference was attributed entirely to the strongest of the native reflections (Zaccai’ & Gilmore, 1979); in the 2nd, the intensity difference was divided according to the ratio of the native reflections (Engelman & Zaccai’, 1980). The main features of the difference Fourier maps did not depend on the approach chosen. All maps shown in Figs 4 and 5 were calculated by attributing the difference to the strongest reflection. All difference Fourier maps were calculated by using the phases of the native structure determined by electron microscopy (Henderson et aE., 1986). Calculation of maps was done by fast Fourier transform methods. The maps and molecular models could be simultaneously displayed on the screen of an Evans & Sutherland model PS300 using the program Frodo (Jones, 1978). Alternatively, molecular models and contours of the electron diffraction map were examinedon Spectragraphics display using the program GACRASTRR written by R. Lavery. Fourier maps of label distributions for different models were calculated by using structure factors corresponding to the *H labels only and plotted to the same resolution as observed data. Model difference Fourier maps were also calculated by treating the powder intensities predicted by a specific model in exactly the same way as the observed intensities; i.e. intensities were scaled and overlapped intensities were split as described above, difference structure factor moduli were calculated by subtracting the native structure factor moduli, using e.m. phases for native PM.

3. Results (a) Sample

preparation

and data collection

Seven new samplesof “hybrid” purple membrane (PM) were prepared (Table 2) and neutron diffraction patterns recorded as described (Popot et al., 1986, 1987; and see Materials and Methods). One sample (657) served as a further control sample, incorporating no ‘H. Two samples (654 and 655) incorporated one bacteriorhodopsin (BR) fragment biosynthetically labeled at the non-exchangeable positions of the leucine residues and the a and /? carbon atoms of the tryptophan residues, reassociated with an unlabeled fragment. The label was either in chymotryptic fragment C-2, encompassing the first two of the predicted transmembrane helices (654), or in fragment C-l, encompassing the rest of the molecule (656) (Fig. 1). In the last four samples (658 to 661), the label was weakened in order to improve the Fourier analysis of the diffraction data. This was done by diluting a labeled fragment with the identical, unlabeled fragment prior to reassociation with the unlabeled complementary fragment. Of those, only sample 660, obtained after threefold dilution of ‘H-labeled fragment C-l and reassociation with unlabeled C-2, exhibited a neutron diffraction pattern significantly different from t,hat of control samples, while the other samples turned out to. be too weakly labeled (see Materials & Methods).

J.-L.

836

Popot et al.

Table 2 Composition of reconstituted samples used in the course of the present work and principal conclusiona drawn from analysie of their neutron diffraction pattern Label in C-17

Sample 267# 2725

-

654

-

655 657 658 659 660 661

La,

DilutionS (mol/mol)

Label in C-2?

bg

WI -

Every position Leu, Trp Trp

NAll 2x

hu, Trp Leu, Trp

3x 3x 6x

Leu, Trp Leu, Trp

Mass BW

Main

conclusion

95 47

Control Either

62

Positions and rotations of A and B Probable position of C Control Control7 Controly Probable position of C Control1

45 17 29 29 29 29

A or B lies in position

78

t Label was in each case on carbon-linked hydrogen positions. In sample 272, every position was labeled (except for the aromatic hydrogen atoms of tryptophan). In the other samples, ‘H was restricted to the leucine carbon atoms and the a and B carbon atoms of tryptophan residues. $ Labeling of samples 658 to 661 was lowered by diluting the labeled fragment with the identical, unlabeled fragment prior to renaturation. Q The preparation, and X-ray and neutron diffraction patterns of samples 267 and 272, and a previous analysis of the neutron data from 272 have been described (Popot et al., 1986; Trewhella et al., 1986). 1) Not applicable. 7 Neutron diffraction patterns of these weakly labeled samples did not differ significantly from controls. The data were used as further control data in the estimate of standard deviations on control reflection intensities.

Table 3 Lorentz-corrected rejection intensities for native PM and four

h,k 191

%O 1,2+2,1 330

22

1,3+3,1 490 3,2+2,3 4,1+1,4 5,O 4,2+2,4 5,1+.1,5 4,3+3,4 5,2+2,5 6,1+1,6 7,0+5,3+3,5 6,2+2,6 7,1+1,7 x.2 Residual

Native PM 36695 14512 9434 640 7078 8212 5474 5106 12265 6266 10859 4176 34886 8665 4365 6942 1022 6023

267

Reconstituted 654

reconstituted samples

samples 655

660

Relative S.D. (%I 5 7 8 38 14 14 4 8 11 7 6 14 3 33 14 13 71 25

37006 13480 8456 977 7504 8458 5050 5898 12452 7301 11148 4476 33747 7096 3674 8430 2020 5386

39996 14087 12808 2391 11917 5317 4274 6240 10868 6548 12148 5695 30320 6375 1866 7024 1205 3477

65211 30243 4925 1184 8844 9974 8635 2044 7542 1720 5606 2537 16170 3896 3319 5454 2506 2749

47570 17658 7533 1650 9565 8829 6992 5102 10204 4925 8589 3115 26646 7276 3780 6957 1340 4827

O-69 0.07

536 0.20

46.41 0.58

724 0.22

Average relative 8.~. values were determined been normalized to sum to the same value. described in Materials and Methods, w&4,,, - ~PMII%f

as described in Materials and Methods. Intensities have xf (reconstituted samples vereua PM) was calculated as with v = N - 1. The residual was calculated as

Path of the Polypeptide

Chain in Bacteriorhodopsin

-I

(2 sin &Xl2

Figure 2. Ratio of reflection intensities (sample/control) for 4 of the recotistituted samples used in this study. (0) Sample 267, reconstituted, not labeled, versus native PM; (w) sample 654, 2H-labeled in fragment C-2, versus sample 267; (+) and (0) samples 655 and 660, respectively, ‘H-labeled to various extents in fragment C-l, versus sample 267. The error bars are equal to f (S.D.i ,/2)/Zi, where S.D.i is the average standard error on measurements of the native intensity Ii of reflection i (see Materials and Methods). (b) Locating he&es A and B The nomenclature for the seven helices of the three-dimensional electron density map (Henderson & Unwin, 1975) is that of Engelman et al. (1980; see Fig. 6). Putative transmembrane segments are called helices A to G, in their order in the sequence (Fig. l(a)). Models that define the correspondence between transmembrane helices and putative transmembrane segments are referred to as assignment models. A given assignment model comprises a family of “rotational” variants, that differ in the rotation of the helices around their axes and therefore in the actual position of the amino acid side-chains in the crystallographic unit cell. (i) Fourier maps Powder pattern peak intensities collected from a control, unlabeled sample (267) and from sample 654 (‘H-labeled in helices A and B) are shown in Table 3, together with the associated S.D. values (the determination of S.D. is described in Materials and Methods). Helix A contained 66 ‘H-labeled positions and helix B 40 or 50, depending on its exact position in the sequence (see Fig. 1 and Materials and Methods). The x,” calculated between sample 654 and control was 54, indicating that differences were highly significant (P < 0901). Intensities for seven or eight out of 22 measurable reflections differed from control values by more than ,/2 S.D. (Fig. 2). Difference Fourier maps were constructed using neutron diffraction intensity differences; e.m. data were used for phases as well as for the relative intensities of overlapped reflections (see Jubb et al., 1984). These maps were dominated by a strong peak located in the vicinity of helix 1, towards the peri-

837

meter of the BR molecule, with secondary peaks in the regions of helices 7 and 3 (Fig. 4(a)). In a previous work, we concluded that helices A and B occupy either positions 1 + 7 or positions 6 + 7. The two helices could not be distinguished one from another. Positions 4 + 7 or 5 -I- 7 could not be rigorously excluded, but appeared to be lesslikely on the basis of the diffraction data and from a structural point of view (Trewhella et al., 1986). Among these models, Fourier maps are best consistent with A and B occupying positions 1 + 7. As helix A is more heavily labeled than helix B, the map would tend to suggest that it occupies position 1, with helix B responsible for the secondary peaks near position 7. On the basis of this qualitative analysis alone, the latter conclusion should be regarded as highly preliminary. More systematic and quantifiable results were obtained with the model-building approach. (ii) Model-building analysis (ii)(a) Sample 654 The 42 possible assignment models for helices A and B were tested using the model-building approach. The rotational positions of each of the labeled helices were varied in 60” steps, yielding 36 rotational variants for each assignment model. The quality of the prediction of experimental data was assessedusing a standard x2 test. The assumptions involved in building the models and calculating the predicted intensities were varied to cover every reasonable source of error (see Materials and Methods): namely, the SCALE factor, that takes into account the extent of ‘H labeling as well as the possible contribution of unmodeled parts of the molecule to the diffraction pattern, was varied between @l and 1; reflections that overlap in the powder pattern were weighted down by adding an extra error of up to 40% of the measured intensity to the experimental error; random errors of the same magnitude as the differences likely to exist between phases in electron and neutron diffraction patterns were added to the phases; resolution was limited to 7.1 A (reflection 7,l) or 8.6 A (reflection 5,2); the low-angle reflection 1,l was omitted from the analysis; only those reflections were included that differed from controls by at least ,/2 S.D. (see Fig. 2); the origins of rotation were varied by f20 by moving the helices up and down the sequenceby one residue; the position of helix B was moved down the sequenceby up to five residues, so as to include the ten ‘H atoms of Leu66 in the model. Over 100 searches were performed on the data from sample 654. The results of three of them, including those with the most reasonable sets of assumptions, are summarized in Table 4. Among the eight possible models compatible with the previous assignments (Trewhella et al., 1986), a model in which helix A occupies position 1 and helix B position 7 (model AlB7) consistently gave the best fit to the experimental data under all of the above assumfitions. On the other hand, some of the models

838

J.-L. Popot et al.

Table 4

x,” values A

1

for

the 42 assignment wmdels for

helices

A and B (sample 654)

2

3

4

5

6

7

305 256 1.97

232 1.45 164

1.65 0.85 015

323 1.71 1.67

154 162

1.61 210

B

1

Models are defined by the assignment of helices A (columns) and B (rows) to positions in the structural map. Thick frames (m) indicate models that, are compatible with our earlier analysis using fully *H-labeled C-2 (Trewhella et al., 1986), double-lined frames (7) models that the earlier analysis only marginally rejected. Reflections from 1 ,l to 7,l were included m the analysis. Helix A was made to cover Pro8 to Met32, and helix B to cover Lys46 to Tyr64. In each case, the xz figure shown is that yielded by the best out of 36 rotational variants of eaoh assignment model (66” rotations of each helix). The 3 figures in each cell were obtained under 3 different sets of assumptions. The SCALE factor, which defines the way calculated structure factors for the label are added to structure factors for native PM, was always 66. Top figure, no downweighting of overlapping reflections. Middle figure, the weight of overlapped reflections in the calculation of xz was decreased by increasing the standard error S.D+ on the intensity I, of these reflections by 62 x I,. Bottom figure, overlapped reflections were identically downweighted and, in addition, the impact of errors on native phases was estimated by adding random phase errors of the same magnitude as the differences expected between phases in neutron and electron diffraction patterns. See Materials and Methods for details of these and other tests.

that had been previously rejected occasionally gave fits that were as good if not better than the AlB7 model. This was often the case of models with helix A in position 1 and helix B in either position 4 or 5 (Table 4). This is not too surprising, given that the label is located mainly in the side-chains. Particularly when ‘H atoms tend to cluster on one side of the helix, as is the case in helix B (see Fig. l(b)), relatively similar distributions can be obtained by placing the model helix in either of two neighboring positions and rotating it appropriately. (Compare, for instance, the distribution of H in Fig. 4(b) (B in 7) and 4(f) (B in 5).) Such models cannot necessarily be ruled out on the basis of a single set of data, particularly when, as was the case with sample 654, the number of reflections that differ significantly from native PM is limited.

A preferred rotational orientation was observed for each of the two helices, with a particularly strong constraint on the orientation of helix A (Fig. 3(b) and (c)). This was not unexpected, given the lateral position of the main peak observed in Fourier maps. Tests confirmed that helix A dominated the searches both by virtue of its heavier labeling and because of its position in the structure. The rotational position predicted for helix B depended on that attributed to helix A. When helix A was misplaced, fits could be improved by misplacing helix B in a compensatory fashion, while misplacing helix B had little influence on the optimal rotation found for helix A (Fig. 3(a)). When helix A was set in its optimal position, rotational positions of helix B giving almost equally good fits to the data covered an angular range of about 120

Path of the Polypeptide

Rototional

position

839

Chain in Baeteriorhodopsin

of helix

13 (deg.;

arbitrary

@rigin)

(0)

4 3 “2 2 I 0

I

24 0

280

Rotational

I 320

0 position

40

00

of helix

120

A (deg.;orbitrory

160

200

240

origin)

(b)

0

C’S”““““‘,“, t’s”““““‘,“,

20

0

Rotational

40

00 position

120

160

of helix

200

240

B (deg.; arbitrary

280

3:

origin)

Cc)

Figure 3. Effect of rotating helices A (in position 1) and B (in position 7) on the value of II,‘. Assumptions were the same as for Table 4 (top figure in each cell). (a) Helix A was rotated by 20” increments (vertically), helix B by 60” increments (horizontally). Isoprobability contours go from x,’ = 1 to xf = 16 by increments of 1. The area where x,’ is greater than 16 is shaded. The star indicates the combination of rotations giving the best model. (b) Same analysis, under ) same as in (a); (---) same as for middle lines in Table 4 (downweighting of 2 different sets of assumptions; (overlapped reflections); the x: value shown for each rotational position of A refers to the best of 6 models differing by the rotational position of B (60” steps). The position chosen to build the atomic model of Fig. 6 is shown by an arrowhead. (c) Helix A was held fixed in its optimal position as determined in (b) and helix B rotated by 20” steps. Assumptions are the same as in (b). The position chosen to build the atomic model of Fig. 6 is shown by an arrowhead.

(Fig. 3(c)). There are a number of reasons to consider the rotational assignment of helix B as more tentative than that of helix A. (1) Errors in modeling helix A may influence the determination of the rotational position of helix B, as observed

when A is deliberately misoriented. (2) The exact limits of helix B in the sequence are somewhat uncertain. We checked that the assignment of B to position 7 does not depend on including Leu66 in the model. When this is done, the best orientation

840

J.-L. Popot et al.

for B does not change. Since the position of Leu66 with respect to helix B is not known however, there is some uncertainty regarding the way its contribution to the diffraction pattern (if any) can bias the rotational search. (3) It is not known either to what extent the presence of Pro50 may affect the structure of helix B, and therefore the relative positions of the residues in projection (see Discussion). (ii)(b) Samples 655 and 656 Our assignment for helices A and B was further checked by model-building analysis of the data from samples 655 and 660. These two samples were ‘H-labeled to different extents, in helices C to G. Peak intensity data are shown in Table 3. x,’ values for the comparison of these two samples with control were 46 and 7.2, respectively, reflecting the high significance of the intensity differences. In these samples, A and B are the only unlabeled helices and appear as “holes” in the distribution of ‘H. No information can be inferred about their identity or rotational orientation. In an initial search, all possible assignment models were considered, with every helix (but A and B) being rotated in 120” steps, so as to cut down on computing time. The effect of such a coarser screening is to increase randomly the ~3 for the best rotational variant of each assignment model, blurring differences between assignment models. The xy’ for the correct assignment model is expected, however, to remain within about 1 from the lowest xy” obtained in a given search (see Materials and Methods). In the coarse examination of sample 655, the best x,’ values were obtained for models with one unlabeled helix in position 1. The best x,2 for model AlB7 exceeded the best overall xf by less than 1 (not shown). Taken together with the conclusions from our earlier work (Trewhella et al., 1986), that led to either helix A or B being placed in position 7,

these results in themselves would have suggested that these two helices occupy positions I +7, as concluded independently from the analysis of sample 654. All models that included one unlabeled helix in either position 1 to 7 were further examined in 60” steps. Again, among all models placing one unlabeled helix in position 7, the data from both samples 655 and 660 were best fit when placing the second one in position 1 (not shown). On the other hand, discrimination among models that placed one unlabeled helix in position 1 was poor. For reasons to be given below, it was estimated that the limited accuracy of the present atomic model of BR did not permit a detailed analysis of these two samples. (ii) (c) Sample 272 Finally, we applied the present model-building and statistical analysis to the data collected from sam le 272, in which fragment C-2 had been per- PH-labeled (Popot et al., 1986; Trewhella et a2.: 1986). All reflections from 1 ,l to 7,1 were used, SCALE was varied from 66 to I, and overlapped reflections were assigned an extra error of either 0 or 20%. In all cases, no model gave a xy” value lower than 4 unless one of the ‘H-labeled helices occupied position 7. Furthermore, a clear distinction was now observed in favor of placing the second helix in position 1 (Table 5). As previously found in the analysis of sample 654, a preference for placing helix A rather than helix B in position 1 was found in all calculations, although not always as clearly as in the search shown in Table 5. As far as sample 272 is concerned, this preference is not very meaningful, as the labeling of the two helices was not very different and the search may have been affected by the ‘H present in the extramembrane loops and tails of fragment C-2.

Table 5 x,’ values for the 42 assignment

Neutron (Trewhella evaluation. overlapped

models for he&es A and B (sample 272)

d&action data collected from sample 272, in which fragment C-2 w&9 per-*N-labeled et al., 198f3), were reanalyzed following the present procedures for model building and See the legend to Table 4. In this particular search, SCALE factor was 03 and errors on reflections were increased by O-2 I,.

Path of the Polypeptide (iii) Predicted Fourier maps Conclusions from the model-building analysis were further examined by comparing predicted distributions of ‘H and predicted difference Fourier maps with experimental observations. Figure 4(b) shows the ‘H scattering-length density distribution at 8.6 A resolution, as calculated from the best AlB7 rotational variant, and the difference Fourier map calculated from the predicted intensities. The main peak of scattering-length density overlaps with the major experimental difference peak (Fig. 4(a)). The four peaks of the experimental map are relatively well predicted from the model, including the artefactual peak near helix 3. The marked differences between the difference Fourier maps and the actual distribution of ‘H around position 7, on the other hand, is illustrative of the difficulty one would face in interpreting the map without the help of model building. Similar comparisons were done systematically for (1) all models that gave reasonably good fits to the experimental data, even though neither the A or B helix was predicted to lie in position 7; (2) the best rotational variant of all models with either the A or B helix in position 7; (3) a selection of rotational variants of the AlB7 model. This study, four representative examples of which are shown in Figure 4(c) to (f), confirmed that the best model according to the model-building search gave also the best fit to the experimental map. (c) Constraints

on the position of helix C

(i) Fourier maps As noted above, two of the samples labeled in helices C to G (samples 655 and 660) yielded diffraction patterns that differed significantly from native atterns (Table 3 and Fig. 2). Both samples were PH-labeled at the same positions, but the level of labeling was threefold higher in sample 655. Due to the uneven distribution of leucine and tryptophan residues, the most strongly labeled helix in these samples is helix C (Fig. 1). The difference Fourier maps for both samples are shown in Figure 5. Contour levels in the difference maps were set to be threefold larger for sample 655 as compared to sample 600. A similar density distribution was expected, therefore, and is in fact observed. The label appears to be distributed towards the center of the molecule, with the highest density peak between helices 6 and 7. (ii) Model-building analysis As indicated above, model-building analysis of the data from sample 655, allowing all helix permutations, was consistent with helices A and B occupying positions 1+7. These helices were constrained to these positions and a complete search of all remaining permutation models was done, allowing for all combinations resulting from 60” rotation for each ‘H-labeled helix. This search indicated that no good fit could be obtained unless helix C was placed in either position 3 or 6, with position

Chain in Bacteriorhodopsin

841

6 being marginally preferable (Table 6). The xz values, however, were much higher than previously observed with sample 654. Even the best were higher than 1.6 (5% confidence level), indicating that the prediction of intensities was poor. This is probably due to two effects. First, a screening finer than 60” was not attempted, given the large number of helices to be rotated; second, the atomic model is approximate (see Materials and Methods) and the heavier labeling of sample 655 makes the contribution of errors in model building felt more strongly in the overall intensities. While not unexpected, these effects cast some doubt on the possibility of discriminating among the nearly lo6 possible rotational variants of the 120 remaining assignment models with the same reliability as observed with the mere 1512 variants to be examined in the case of sample 654. For this reason, it was felt that a detailed analysis of the data collected from samples 655 and 660 would have to be deferred until a better structural model of BR could be developed and tested. While placing helix C in position 6 would appear most compatible with both the Fourier maps and the model-building data, we think that at this point such an assignment should be regarded as tentative.

4. Discussion (a) Assignment

of he&ices A and B

In spite of many attempts to do so, the assignment of BR sequence segments to structural map densities has met with little success. One difficulty has been that the elements of structure that have been localized (e.g. retinal (Jubb et al., 1984; Seiff et al., 1985) or the extramembrane C-terminal end of the protein (Wallace & Henderson, 1982)) may not lie close to the axis of the u-helix to which they attach. Certain folding models have been excluded, but hitherto none of the seven transmembrane helices has been assigned to a given sequence segment. In our previous work with hybrid PM (Popot et al., 1986; Trewhella et al., 1986), the entire C-2 fragment was labeled isotopically, including the extramembrane loop and tails, with the label being almost equally strong in both helices A and B. Using our earlier analytical approach, we established the end of the protein where these two helices lie, but we were not able -to assign each of them to a defined position in the structure. The present work unequivocally yields the assignment of the first two transmembrane segments and puts restrictions on the position of the third. The sample labeled selectively at non-exchangeable positions in the leucine and tryptophan residues of the C-2 fragment permitted us to locate helices A and B. The assignment of one of these helices to position 1 in the structural map was indicated both by model building and by the difference Fourier map. The assignment of the second one to position 7 was compatible with both approaches. A few alternative assignments could not be defi-

842

J.-L.

Popot et al.

Figure 4. Observed and predicted difference Fourier maps for sample 654 (‘H-labeled in fragment C-2). The same arbitrary units (a.u.) have been used for contour levels throughout the Figure. (a) The difference Fourier projection map of sample 654. The outline of the unlabeled BR trimer, in blue, was plotted by using neutron diffraction intensities and electron diffraction phases and powder ratios (see Materials and Methods). Contours shown are at 500 and 1500 a.u. Helix positions are numbered according to Engelman et aZ. (1980). Data (from reflection 1,l to 5,2) from sample 654 were scaled to the same sum of intentities as native data. For overlapped reflections, the difference was assigned to the strongest (see Materials and Methods). The difference Fourier map is shown in red. Contours are at 100, 200, 300 and 400 a.u. (b) to (f) A comparison of the 2H distribution (yellow) and predicted difference Fourier maps (red) for different models, shown at the same resolution as in (a). Difference Fourier maps were calculated from predicted intensities following the same procedure as in (a) see Materials and Methods). (b) Helix A was placed in position 1, helix B in position 7. Rotations are those giving the best fit to observed reflection intensities (see Figs 3 and 6). Under the set of assumptions used in

Path of the Polypeptide

Chain

Table 6 Assignment of helix C based on the model-building analysis of diffraction intensities for sample 655 Assignment of helix C

Best reduced

to position 2 3 4 5 6

1’ in calculation

I

II

III

3-61 1.74 2.53 2.87 1.68

345 221 390 311 2.04

323 200 2.83 3-03 1.75

All models had helices A and B in positions 1 and 7. Each of the remaining 120 assignment models has 15,625 rotational vatiants generated by the combination of 6OO” stepwise rotations of the C to G helices. The Table lists the xz for the best model for each positional assignment of helix C, under 3 sets of assumptions. Overlapped reflections were downweighted as described in the legend to Table 4. I, SCALE factor 0.6, reflections 1,l to 5,2; II, SCALE factor @6, reflections 1.1 to 7,l; III, SCALE factor 0.7, reflections 1.1 to 7,l.

in Bacteriorhodopsin

843

The observation that difference Fourier maps, independent from any model building, assignpart of the C-2 fragment to position 1 is a strong confirmation that the electron density in this region of the map must be interpreted as an u-helix (Henderson & Unwin, 1975; Tsygannik & Baldwin, 1987) and not as two /?-strands, as has been proposed (Jap et al., 1983). Spectroscopic data that have been taken to indicate the presenceof transmembrane /&strands in BR certainly must be interpreted in some other manner. Besides our previous work, there are few data available regarding the position of helices A or Bin the structural map. Seiff et al. (19866) labeled lysine 41 with ‘H-labeled phenylisothiocyanate (D,-PITC). In the usual topological model of BR (Fig. l(a)), Lys41 is located at the N-terminal extremity of helix mined, by neutron

B. Seiff and co-workers diffraction, the position

center of gravity of D,-PITC ture,

15 to

17 A off the

deterof the

in the projected strucaxis

of helix

7. They

concluded that helix B could not be in position 1 or in position 7. However, the combined length of the nitely ruled out on the basis of the present data alone, but they all would be incompatible with the strongest conclusion from our previous work: namely, that either A or B lies in position 7 (Trewhella et al., 1986). In order to check on this earlier conclusion, we reanalyzed the experimental data from the sample containing per-2H-labeled C-2 (Trewhella et al., 1986) using the present approaches to model-building and statistical analysis. This re-examination confirmed the assignment of either helix A or B to position 7. It further showed that models with the second helix in position 1 predicted these earlier experimental data much better than any other, in agreement with the new data gathered from the selectively 2H-labeled sample. The Fourier map,calculated from the new experimental data suggested that the most heavily labeled segment, helix A, lies in position 1. This assignment was strongly supported by the model-building analysis. The experimental difference maps were indeed much better predicted by assigning A to 1 and B to 7 than by the reverse assignment. We have previously shown that the isolated fragment C-2, refolded in lipid vesicles, takes up a highly

a-helical

structure.

Upon

fusion

with

vesicles

containing C-l, the two fragments reassociate and reform BR, with minimal change of their a-helix content (Popot et al., 1987; Popot & Engelman, 1988 and unpublished results). Therefore, C-2 also must be primarily a-helical when it is part of BR.

lysine

side-chain

and the label measures

about

10 d.

The position of the C” of Lysdl with respect to the axis of helix B is itself uncertain, as this residue lies at one end of the helix. Ovchinnikov et al. (1985) have reported that, in native PM, Phe42 is accessible to monoclonal antibodies, which suggests that helix B starts farther down the sequence. Two residues in extended configuration would allow D,-PITC to lie in the position located by Seiff et al. (1986b). More recently, Kouyama et al. (1988) labeled Lys41 with a fluorescent derivative. Fluorescence energy transfer and fluorescence depolarization measurements suggestedthat the label lay in the vicinity of position 7, in better agreement with the conclusions from the present work. It is not known to what extent the nature of the group attached to the lysine residue might affect the conformation of the loop between transmembrane segments A and B, which could account for these seemingly conflicting results. (b) Rotational

positioning

Models that differed by the rotational positions of A and B around their axes were compared using the model-building approach. Rotating helix A had the most impact on such searches. The rotational position giving the best fit to the experimental data had Gly16 turned towards the middle of the BR molecule (Fig. 6). Predicted difference maps fitted

Table 4 (top line in each cell). xt =094. Yellow contours at 285 and 570 a.u. Red contours at 130, 260, 390 and 520 a.u. (c) Same assignment the rotational and 406 a.u.

(x:=2.65).

as in (b), but helix A was rotated

position (d) Helix

in (b) and helix B was allowed to relax to red

contours model for

at 100, 200, 300 this assignment

contours at 375 and 750 a.u., red contours at 80, 160, 240 and 320 a.u. (e) Helix A in position 1, helix is the best rotational model for this assignment (xz = 1.13). Yellow contours at 315 and 630 a.u., red contours at 150, 3od, 450 and 600 a.u. (f) Helix A in position 1, helix B in position 5. This is the best rotational model for this assignment (x,‘=2.03). Yellow contours at 300 and 600 a.u., red contours at 180, 360, 540 and 720 a.u. B in position

Yellow

by 180” from its position

giving the lowest xt (6.54). Yellow contours at 675 and 1350 a.u., A in position 7, helix B in position 1. This is the best rotational

4. This

(a) Figure 5. Difference Fourier maps (red) for samples (a) 655 and (b) 660 (‘H-labeled

(b)

in fragment C-l). The contours of the native trimer &t 500 and 1500 arbitrary units (a.u.) are shown in blue. Difference Fourier maps were calculated as for sample 654, except that the intensities were scaled to the sum of native intensities multiplied by 1.30 (sample 655) or 1.10 (sample 660) (see Materials and Methods). Since sample 660 contains l/3 the amount of ‘H as sample 655, difference Fourier contours for sample 655 (228, 456 and 684 a.u.) were chosen to be 3 times larger than those for sample 660 (76, 152 and 226 a.u.).

Figure 6. Ribbon representation of helices A a\d B in their optimal positions, with selected side-chains. Note that the rotational position of helix A was more constrained by the data than that of helix B (see Fig. 3 and the text). Side-chains are in the extended conformations described by Diamond (1979) and are colored according to their probable depth in the tier. Helix positions are indicated by membrane: red, outermost tier; yellow, central region; white, innermost superimposing selected contours from the 3-dimensional map calculated by Tsygannik & Baldwin (1987). The approximate positions in projection of the retinal (Jubb et al., 1984) and Schiff base (SB; Seiff et al., 1986a; Heyn et al., 1988) are shown. The green oblique bar is 5 A long. The Figure was prepared using programs written by R. Lavery (Institut de Biologie Physico-Chimique, Paris).

Figure 7. A sketch showing the areas of helices A and B that face various parts of BR and of its environment. The environment of each side-chain in the best atomic model was examined using molecular graphics. Areas of interaction were mapped on helix nets and approximated by vertical stripes. The generatrix of each helix that faces the approximate direction in which the Schiff base lies is indicated by a red line.

Path of the Polypeptide

Chain in Bacteriorhodopsin

best with the experimental map when helix A was rotated as indicated by model building. Examination of molecular models shows that in this orientation strongly hydrophobic residues, leucine residues in particular, face the lipids that surround the BR trimer, as sketched in Figure 7. Less hydrophobic residues tend to face the inside of the molecule (Gly16, Thrl7, Gly23 and Thr24). This distribution is consistent with early indications that BR might be an “inside-out” protein, in the sense that its constitutive helices would expose their most polar face toward the inside of the molecule (Engelman & Zaccai, 1980). This view has also been supported by labeling studies of helix C using a lipophilic reagent (Brunner et al., 1985) and by sitedirected mutagenesis (Khorana, 1988). The sketch in Figure 7 also indicates which regions of helix A face helices 2 and 7. The residue of helix A whose C” lies closest to helix 7 is probably Met20. The rotational position of helix B had a weaker influence on xz and could be defined only to within +60”. When helix B is set in the middle of this range, Thr46 faces the middle of BR (Fig. 6). This conclusion should be regarded as, more tentative than that regarding helix A (see Results). In particular, it is possible that the presence of Pro50 induces a stagger between the two halves of the helix (see below). If such were the case, the orientation indicated by our data would be some average between that of the outward-facing and inwardfacing parts of the helix. Assuming the helix not to be severely disturbed by the proline residue, its orientation would be such as to place a number of strongly hydrophobic residues facing the lipids in the center of the BR trimer (Fig. 7). Thr55 and Ser59 would be close to helix 4 of the neighboring BR molecule, perhaps in a position to form hydrogen bridges with it, stabilizing the BR trimer. Pro50 would be turned toward the middle of BR, facing helix 2, close to the point where helices 1 and 7 come in closest contact. The demonstration that helix 7 contains a proline residue is an interesting aspect of the present work. Proline cannot be incorporated in an undistorted cc-helix because of steric hindrance (e.g. see Piela et al., 1987). In soluble proteins, proline residues are generally accommodated into a-helices by changes of direction of the helix axis that reach typically 20” to 30”; broken main-chain hydrogen bonds are systematically reformed with water or with the protein (Barlow & Thornton, 1988). Inside membranes, the absence of water is likely to increase the energetic cost of kinks and this could tend to limit their amplitude. Such does not seem to be the case for helix C of the L subunit of Rhodopseudomonas viridis reaction center, which contains a proline residue in position 124 (Michel et al., 1986) and appears bent (Deisenhofer et al., 1984). In the structural map of BR, on the other hand, helix 7 appears almost straight (Tsygannik & Baldwin, 1987). Local distortions (perhaps involving 310 structure) may allow the helix to maintain an essentially straight course, as observed

845

in one of the conformations of alamethycin (Fox & Richards, 1982). Genetically engineered replacement of Pro50 with alanine yielded active BR with a spectrum almost identical with that of the native molecule (Khorana, 1988), which is consistent with the view that Pro50 does not severely disturb the helix. It is likely in any case that Pro50 perturbs the hydrogen-bonding pattern and it may introduce some stagger between the two halves of the helix. This point deserves further investigation. It is worth noting that, in the tentative orientation shown in Figure 6, the carbonyl group of Thr46, that would form a hydrogen bond with the amino group of residue 50 were it not proline, faces the inside of BR. Tyr43, Tyr57 and Tyr64 lie on the same helix face. They would also be turned toward the center of the molecule, facing helix 2, close to the surface of helix 1 (Fig. 6). Each tyrosine residue in BR can be substituted with phenylalanine without impairing proton pumping (Mogi et al., 1987). Replacement of Tyr43 and Tyr64 had no effect on the spectrum. Replacement of Tyr57, on the other hand, yielded a slightly blue-shifted BR, unstable in detergents, and exhibiting a markedly slower rate of renaturation (Mogi et al., 1987). According to our results, it is conceivable that Tyr57 stabilizes the association of helices A and B by hydrogen bonding either to the polypeptide backbone of helix 1 or to Thr17. Its substitution by phenylalanine would loosen this association. This could diminish the stability of the BR molecule, slow its renaturation and, indirectly, affect the spectrum of the chromophore. Whatever the orientation given to helix B, the three tyrosine residues in this helix, as well as Tyr26 in helix A, are all too remote from the retinal to correspond to the tyrosinate residue that interacts with the positive charge on the Schiff base in lightadapted BR (Roepe et al., 1988). This is consistent with the recent asignment of the corresponding infrared signal to Tyr185 (Braiman et al., 1988a). Altogether, the orientations of helices A and B as deduced from the neutron diffraction data are close to those proposed on the basis of helix lateral amphipathy and site-directed mutagenesis experiments (Mogi et al., 1988; Khorana, 1988). (c) Assignment

of helix C

The two samples labeled in the C-l fragment gave some indications about the possible position of helix C, the most strongly labeled helix in this fragment. Model building suggested that helix C lies in either position 6 or position 3. Difference Fourier maps favored position 6. This conclusion should be regarded as more tentative than that concerning helices A and B as none of the predictions was very good according to our xz test, perhaps a result of limitations in the atomic model. Recently, the loop between helix B and helix C has been shortened by genetic deletion of 11 residues, Although their properties became temperature-dependent, the modified BR molecules folded, bound retinal and

J.-L. Popot et al.

846

pumped protons (Gillies-Gonzales, 1988). These observations are also most consistent with assigning helix C to position 6, next to helix B. If helix C lies in position 6, the lipophilic labeling data of Brunner et al. (1985) would define a rotational orientation such that Asp85, Thr89 and Asp96 face the position of the retinal (Jubb et al., 1984; Fig. 6). A contribution of these residues to the environment of the retinal is strongly indicated by site-directed mutagenesis (Mogi et al., 1988; Khorana, 1988). The protonation of these two aspartate residues changes during proton pumping (Braiman et al., 19883) and their substitution by asparagine abolishes pumping more or less completely (Mogi et al., 1988; see also Marinetti et al., 1989; Holz et al., 1989; Butt et al., 1989). Substitution of Thr89 by valine dramatically affects the spectrum of BR (Khorana, 1988; seenote added in proof). (d) Overall

model of BR

Our present conclusions regarding helices A and B are in agreement with a model first proposed by Engelman et al. (1980), according to which transmembrane segments follow each other without interleaving, starting with helix A in position 1 and running clockwise in the projection used here. Our tentative assignment of helix C to position 6 is also in agreement with this model. From their experiments with partially *H-labeled retinal, Heyn and co-workers concluded that helix G, to which the retinal is bound, lies in either position 2 or 6 (Seiff et al., 1986a; Heyn et al., 1988). Placing helix C in position 6 would constrain helix G to lie in position 2, again in accordance with the model. This assignment for G is compatible with the data of Wallace & Henderson (1982) on the location of the C-terminal tail of BR, with those of Katre et al. (1984) on the position of a label attached to Arg225 and Arg227 (at the end of helix G) and with the observation by Dencher et al. (1988) of a small conformation change in the vicinity of position 2 accompanying the BR,,, to M transition, It contradicts one of the conclusions of Trewhella et a2. (1983), placing G in position 4. The difficulties inherent in the earlier methods of labeling and analysis, however, make it probably desirable to re-examine this latter conclusion using the present constraints on the position of A and B, and a more sophisticated model-building procedure. Covalent labeling with a photoreactive retinal analog led Huang et al. (1982) to conclude that helix F lies side-by-side with helix G. This conclusion was recently further reinforced by the identification of Tyr185, in helix F, as the tyrosinate that interacts with the Schiff base (Braiman et al., 1988a). When A, B, C and G have been placed as described above, this implies that F lies in position 3, as predicted by the Engelman et al. (1980) model (and concluded by Trewhella et al., 1983). The relatively short length of the loop between helices C and D (seeFig. 1) suggests that D lies close to C, i.e. in position 5, leaving position 4 as the only remaining assignment for helix E. A model without inter-

leaving is consistent with recent genetic evidence showing that two extramembrane loops in BR can be shortened (Gillies-Gonzales, 1988). It is also in keeping with the most frequent arrangement of helical bundles in soluble proteins (e.g. see Finkelstein & Ptitsyn, 1987, and references therein). One should recall, however, that a limited interleaving is observed in the structure of bacterial photoreaction centers (Deisenhofer et al., 1984; Allen et al., 1987). While the 1980 model now appears most likely to be correct, some assignments remain tentative. In the recent past, experimental results have often been interpreted on the basis of this model. This has been the case with analyses of site-directed mutagenesis experiments (Khorana, 1988; Mogi et al., 1988; Braiman et al., 198%; and references therein) and with comparisons of halorhodopsin and BR structures (Lanyi et al., 1988; Schobert et aZ., 1988; Oesterhelt & Tittor, 1989). The present work, together with the speculations outlined in the above paragraph, puts these interpretations on a more secure footing, even though it does not uniquely establish the model. It should be of help in defining the environment of the Schiff base and the mechanism of proton pumping. The main conclusion of our study, namely the determination of the positions and orientations of helices A and B, should form a useful basis for further investigations into the structure of bacteriorhodopsin.

We thank: R. Henderson and J. Baldwin (MRC, Cambridge) for communication of electron microscopy data prior to publication; S.-E. Gerchman (Brookhaven National Laboratory) for her expert help in preparing the BR fragments; P. D. McCrea and T. R. Kahn (Yale University) for their help in overcoming the practical difficulties of long-distance collaboration; G. Davis and A. J. Lanzetti (Yale) for amino acid analyses; M. Stromski (Yale) for preliminary n.m.r. data on the purified BR fragments; C. Kipnis (Universite Paris IX) for a discussion on statistics; B. Pullman (IBPC, Paris) for access to the Vax computer; R. Lavery, 8. Furois-Corbin. D. Piazzola and R. Savinelli (IBPC) for their very kind help with computers and molecular graphics; M. LeBars (Universite VI, .Paris) for communication of her X-ray data on trichorzianin; F. Bon (Institut Jacques Monod. Paris) for preparing Fig. 3(a); Y. Pierre and G. Esculier (IBPC, Paris), F. Samatey and M. Ferrand (ILL. Grenoble) for their help with putting together the Figures and building balsawood models of BR; A. Gabriel (EMBL Grenoble) for access to microcomputers: J. Smith (Harvard University) for advice and encouragement to one of us (O.G.); J. Deatherage (University of Arizona, Tucson) for his gallant attempts at teaching one of us (J.-L.P.) crystallography; and our colleagues from the Service de Photosynthese and Service de Biochimie Theorique at the Institut de Biologie Physico-Chimique for many fruitful discussions. This work was supported by a joint CNRS-NSF grant to J.-L.P. and D.M.E., a grant, from the Ministire de la Recherche et de la Technologie to J.-L.P., 2 NIH grants (GM22778 and GM39546) and an NSF grant ENT8612425to D.M.E.

Path

qf the Polypeptide

Chain

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