Biophysical Journal Volume 65 September 1993 1295-1306

Two-dimensional Crystallization of Escherichia coli-expressedBacteriorhodopsin and Its D96N Variant: High Resolution StructuralStudies in Projection

Alok K. Mitra,* Larry J. W. Miercke, George J. Turner, Richard F. Shand,t Mary C. Betlach,and Robert M. StroudDepartment of Biochemistry and Biophysics, University of California, San Francisco, California 94143-0448; *Department of BiologicalSciences, Northern Arizona University, Flagstaff, Arizona 86011 USA

ABSTRACT Highly ordered two-dimensional (2-D) crystals of Escherichia cofi-expressed bacteriorhodopsin analog (e-bR) andits D96N variant (e-D96N) reconstituted in Halobacterium halobium lipids have been obtained by starting with the opsin proteinpurified in the denaturing detergent sodium dodecyl sulfate. These crystals embedded in glucose show electron diffraction inprojection to better than 3.0 A at room temperature. This is the first instance that expressed bR or a variant has been crystallizedin 2-D arrays showing such high order. The crystal lattice is homologous to that in wild-type bR (w-bR) in purple membranes(PM) and permit high resolution analyses of the structure of the functionally impaired D96N variant. The e-bR crystal is iso-morphous to that in PM with an overall averaged fractional change of 12.7% (26-3.6-A resolution) in the projection structurefactors. The projection difference Fourier map e-bR-PM at 3.6-A resolution indicates small conformational changes equivalentto movement of -

Volume 65 September 1993

and SB reprotonation (Braiman et al., 1988; Gerwert et al.,1989; Holz et al., 1989; Otto et al., 1989; Thorgeirsson et al.,1991) and is deprotonated during the M -* N transition(Bousche et al., 1991). The formation of the photo-excitedM412 intermediate in the Asp85-Asn variant is inhibited re-sulting in complete extinction of proton pumping (Braimanet al., 1988; Miercke et al., 1991). Interestingly, recent stud-ies on this variant from H. halobium-expressed materialshows that in the pH range 6-12 the protein exists as threespectroscopic species in equilibrium which are homologs ofM, N, and 0 photo-intermediates (Turner et al., 1993). TheD96N variant shows reduced proton pumping due to sloweddecay of the M412 intermediate (Holz et al., 1989). Anotheraspartate, Asp212 has been suggested to play a more indirectrole acting as one of the participants of the proton acceptorcomplex (Needleman et al., 1991).

In functionally impaired variants the effect exerted bysingle amino acid substitution on the photocycle and chro-mophore environment can also be modulated by structuralrearrangements. Therefore, it is necessary to characterize anychange in the structure in addition to function to separatelocalized chemical/electrostatic effect of mutation from pos-sible large-scale structural alterations relayed to the rest ofthe protein. In this work we have used electron diffraction asa sensitive high resolution assay (Mitra and Stroud, 1990) toprobe the structure in the functionally impaired D96N vari-ant. Starting with e-bO expressed in large quantities (Shandet al., 1991) and purified to homogeneity (Miercke et al.,1991) we developed a method for obtaining large two-dimensional (2-D) crystals of e-bR that diffracted to highresolution and have lattices very similar to that in PM. Us-ing an analogous protocol, we prepared 2-D crystals of thee-D96N variant. Here we compare in projection the groundstate structures of e-bR and wild-type bR in PM andthe structures of e-D96N and e-bR by difference Fourieranalyses.

MATERIALS AND METHODS

MaterialsBuffers used were the following: 0.1% (w/v) sodium dodecyl sulfate (SDS;Bio-Rad, Richmond, CA), 100 mM sodium acetate, pH 6.0 (Buffer A); 50mM NG (CalBiochem, San Diego, CA), 5 mM sodium acetate, 4 mM so-dium azide, pH 5.0 (Buffer B); 18 mM NG, 5 mM sodium acetate, 4 mMsodium azide, pH 5.0 (Buffer C); 10 mM sodium acetate, 4 mM sodiumazide, 16 mM CHAPSO (Boehringer Mannheim Biochemicals, Indianapo-lis, IN), pH 5.0 (Buffer D); Buffer D, 0.5 M NaCl (Buffer E); 100 mMsodium acetate, 4 mM sodium azide, pH 5.0 (Buffer F); 30 mM sodiumphosphate, pH 6.0 (Buffer G); 100 mM potassium phosphate, 6 mM OG(Pfanstiel Labs., Waukegan, IL), 200,uM DTAC (Eastman Kodak Co.,Rochester, NY), pH 5.2 (Buffer H); 10 mM sodium acetate, 4 mM sodiumazide, pH 5.0 (Buffer I); 10 mM sodium acetate, pH 5.0 (Buffer J); TritonX-100 (Pierce, Rockford, IL), DMPC (Avanti Polar lipids, Birmingham,AL), CHAPS (Fluka, Ronkonkoma, NY), all-trans retinal (Sigma ChemicalCo., St. Louis, MO). D-Glucose and the salts were reagent grade.

Purification and 2-D crystallization ofE. coli-expressed proteinLarge-scale e-bO expression (17 mgs/liter) was carried out according toShand et al. (1991). Retinylation and purification to homogeneity of wild-

type fusion protein e-bR and e-D96N variant were carried out as described(Miercke et al. 1991) with modifications. All-trans retinal incorporation wascarried out overnight upon mixing, by volume, 200 parts of 0.3-0.4 mg/mlof protein in BufferA, 40 parts of 4% DMPC/4% CHAPS in water, 80 partsof water and 2 molar equivalents (retinal to protein) retinal. The reactionmixture was 0.2-gm filtered, concentrated 4-5-fold with high pressure fil-tration,YM-30 membrane (Amicon Inc., Beverly, MA), and applied (3-5-mlsample/tube) on top of a 6.5% (w/v) sucrose in Buffer B with a 22% w/vcushion and spun in a VTiSO rotor at 45,000 rpm, 4°C for 40 h. Protein atthe sucrose-cushion interphase was directly applied to "Red-A" dye-ligandmatrix (Millipore Corp., Bedford, MA) which had been equilibrated withBuffer C. The matrix was washed with 10 column volumes of Buffer D, andthe protein eluted with Buffer E to produce pure bR monomer in CHAPSOwhich contained

Electron Diffraction Studies on Expressed bR

Specimen preparation and electron diffractionCrystalline specimens were applied, typically 4 ,ul, on a 400-mesh coppergrid covered with a thin carbon film. This film was floated off a freshlycleaved mica sheet on to a clean double-distilled water surface and thengently lowered onto the grids. The carbon-coated grids were air-dried, andthe carbon films were aged for at least 2-3 days to make them hydrophobic.Prior to sample application the carbon film was washed with several dropsof 100% ethanol (Quantum Chemical Corp., Tuscola, IL) and then air-dried.

e-bR crystals suspended at -10 ,uM protein concentration in buffer Icontaining 0.7% (w/v) glucose was used for specimen preparation. e-D96Ncrystals were washed with a 50 X volume of buffer J to remove azide andthen suspended at 10 p.M protein concentration in buffer J containing 0.7%(w/v) glucose for specimen preparation. After letting the sample settle for2 min, excess liquid was drawn off slowly by touching the edge of the gridwith the tip of a cut Whatman filter paper and the grid was air-dried.

H-D96N samples in buffer H were applied at -30 p.M protein concen-tration and allowed to settle for 30s to 1 min. Next the grid was gently tappedon a Whatman filter paper to remove most of the liquid, and then 5 ,ul of0.7% glucose solution in water was applied and allowed to settle for 45 s.Finally, after removing the excess liquid with a cut tip of Whatman filterpaper, the grid was air-dried.

Grids, thus prepared, were examined within 24 h or were stored at -20°Cuntil used.

Electron diffraction from glucose-embedded samples was carried out atroom temperature at 100 kV in an EM400 electron microscope (PhillipsElectronic Instruments, Eindhoven, The Netherlands) using a 30-p.m secondcondenser aperture, 0.2-p.m spot size, and a total dose of -0.4 e/A2. Theprocedure for collecting low dose electron diffraction patterns was as de-scribed by Mitra and Stroud (1990). During each session at the microscope,diffraction patterns from oriented gold crystals (Ted Pella Inc., Redding,CA) and calibration films with known electron exposures were recorded atthe given microscope setting. The [200] 2.04-A reflection from oriented goldcrystals was used as a diffraction calibration standard. The recording filmused was Kodak 4496 (SO-163G) (Eastman Kodak Co., Rochester, NY)which was developed for 16 min in fresh undiluted Kodak D19 developerat 200C.

Data acquisition and analysesDiffraction patterns and films for calibrating optical density to electronswere digitized on a flatbed microdensitometer (model 1010 M, Perkin-Elmer Corp., Norwalk, C`T) whose output was filtered by a 4302 dual 242B/Octave band-pass filter (Ithaco, Ithaca, NY) to damp high frequency noise.The aperture and step size for digitizing the diffraction patterns was 10 ,um,while these were 30 p.m for digitizing the dose-calibration films.

Reconstituted e-bR and e-D96N sheets sometimes consisted of asingle surface lattice but usually had two and up to four superimposed P3lattices each of which were twinned, in general, to different extents. In or-der to visually assess the resolution, specimen tilt and number of overlap-ping lattices each digitized diffraction pattern was displayed on a Parallax1280 display processor (Parallax Corp., Sunnyvale, CA) after 3X3nearest-neighbor pixel averaging. Using the positions of the maxima ofstrong reflections, the least-squares refined [01] and [10] lattice vectorsfor all contributing lattices were obtained. The digitized pattern wassmoothed and optical densities converted to electrons using the data fromcalibration films relating optical density to electron exposure (Katre et al.,1984). Next, starting with the predicted positions of all the reflections inthe lattice that showed most intense spots (major lattice), a "matched fil-ter" algorithm (Katre et al., 1984) was employed to accurately locate thecenters and evaluate the background-corrected intensities. In this processwhenever the center of a reflection spot for this "major" lattice waswithin twice the average spot radius of spot/s belonging to the other "mi-nor" lattice/s it was not considered for intensity measurement. Such rejec-tions usually constituted

Volume 65 September 1993

FIGURE 2 Digitized display of high reso-lution electron diffraction pattern from aglucose-embedded e-D96N crystal after sub-tracting contribution from inelastic scatter. Thepattern is from two major crystalline domainsinclined to each other by an angle of 8°. Dif-fraction spots to 2.9 A are visible. The circulararcs represent 3.1-A resolution.

A. This is 62.5 A in PM as determined by Henderson andcoworkers for native membranes (Henderson and Unwin,1975) or fused sheets (Henderson et al., 1990) at -160°C and61.9 A as determined by Hayward and Stroud (1981) fornative membranes at -120°C. Thus there is -1% increasein the lattice dimension in the lipid-reconstituted crystal ofthe E. coli-expressed protein. The values of Rsym and Rmergefor the data from e-bR and e-D96N crystals for all reflec-tions (26-3.6-A resolution) and in resolution shells are pre-sented in Table 1. In Table 2 the merged projection structurefactors IFNI (for e-bR) and FDI (for e-D96N) and theirstandard deviations together with the projection structurefactors F(PM) are listed.As a function of resolution the profiles of the variations

of IF (PM) and FN I, averaged in resolution shells, arecompared in Fig. 3. The similarity of these profilesthroughout the resolution range is apparent and the averageof the difference amplitude ( AF )(( FN - F(PM) )does not decrease or increase monotonically. This indicatesthat the wild-type crystal in PM and the reconstituted crys-tal of bR analog are isomorphous. Under the assumptionthat the 1.1% increase in unit cell dimension (62.5 to 63.2A) is isotropic and that no molecular reorientation hastaken place in the reconstituted crystal, we estimate a 2.3%fractional change in the structure factor in the resolutionrange considered. This is much smaller than the observedoverall fractional change (II FN - F(PM) 1)/( F(PM) )

TABLE 1 Symmetry and merging R factors* for thediffraction patterns from crystals of e-bR and e-D96N used Insynthesizing difference Fourier maps

e-bR, four patterns e-D96N, five patterns

OnI On IFI OnI On IFI

Rsym*(26.0-7.0 A) 11.5 7.3 9.1 5.8(7.0-5.0 A) 26.7 15.1 22.7 13.2Overall (26.0-3.6 A) 16.8 11.9 12.5 9.1

Rmerge§(26.0-7.0 A) 7.2 4.8 6.2 4.1(7.0-5.0 A) 16.8 10.0 11.8 7.8(26.0-3.6 A) 11.8 9.2 8.1 6.3

*Based on extracted data after correcting for twinning and tilt.tAverage value of the agreement index. This for a given diffraction patterncalculated on the structure factors is expressed as,

n / n

Rsym = E IlFil - (IFI)I / IFihk i=1 hk i=1

§Agreement index for merging of data sets calculated on the structure factorsis expressed as,

N N

Rmerge = E IFjI - ImergeII / 1Fjlhk j= hk j=1

where Fj (= (F I)) is the six-fold symmetry averaged value for a givenreflection in a pattern and Fmerge is the corresponding merged value fromN patterns.

1 298 Biophysical Journal

TABLE 2 Averaged projection structure factors F and their experimentally determined standard deviations r to 3.6-Aresolution. I FN I Is for merged data from e-bR crystals and I FD I Is formerged data from e-D96N crystals.H K IF(PM) I * FN It UNt I FDIt T|D* H K I F(PM) I * I FNI UNt l FD I't rDt1 21 31 41 51 61 71 81 91 101 111 121 131 142 12 22 32 42 52 62 72 82 92 102 112 122 132 143 03 13 23 33 43 53 73 83 93 103 113 123 134 04 14 24 34 44 54 64 74 84 94 104 114 125 05 15 25 35 45 55 65 75 85 95 105 116 0

10.725.58

13.6115.854.5813.474.813.823.503.69

10.238.032.425.4513.778.71

22.976.737.75

10.098.558.176.486.635.452.061.206.70

16.8613.161.64

15.5315.326.344.461.812.357.144.732.86

13.5917.0710.1625.246.645.534.074.783.574.936.822.623.09

20.715.6317.475.552.953.783.776.533.121.612.183.25

12.68

9.90 0.195.49 0.27

14.13 0.2115.38 0.323.23 0.25

12.36 0.094.40 0.133.47 0.154.03 0.194.36 0.088.83 0.378.33 0.532.71 0.596.92 0.39

13.17 0.057.48 0.12

22.46 0.097.16 0.457.51 0.398.73 0.068.64 0.176.96 0.416.18 0.235.59 0.515.58 0.802.18 2.181.92 0.095.44 0.2015.12 0.2912.32 0.493.60 0.2717.17 0.3216.86 0.516.84 0.194.04 0.442.33 0.202.72 0.135.96 0.565.14 0.312.86 0.31

12.26 0.3415.94 0.179.59 0.49

26.93 0.426.71 0.515.87 0.804.62 0.675.23 0.353.79 0.365.04 0.645.93 0.183.77 0.624.04 0.57

19.14 0.185.55 0.50

17.83 0.254.06 1.122.17 2.173.15 0.273.14 1.243.82 0.412.26 0.222.42 0.292.32 0.202.62 0.2611.94 0.36

9.85 0.065.01 0.1212.89 0.0915.93 0.304.24 0.0912.50 0.194.90 0.273.79 0.133.95 0.453.28 0.259.30 0.057.40 0.304.08 0.205.86 0.1713.60 0.097.71 0.14

22.42 0.097.41 0.338.37 0.109.25 0.168.54 0.137.43 0.156.67 0.106.52 0.244.92 0.242.47 0.211.54 0.204.78 0.12

16.13 0.1213.75 0.334.16 0.2817.79 0.3115.86 0.147.18 0.264.26 0.132.49 0.132.61 0.036.68 0.174.42 0.192.27 0.13

12.59 0.0615.56 0.289.33 0.03

26.25 0.416.63 0.276.28 0.183.04 0.494.88 0.033.99 0.255.96 0.145.98 0.103.36 0.182.59 0.16

19.26 0.194.78 0.4517.88 0.284.17 0.193.12 0.112.78 0.053.12 0.054.59 0.172.26 0.101.80 0.121.87 0.122.95 0.14

12.22 0.20

6 16 26 46 56 66 76 86 96 106 117 07 17 27 37 47 57 67 77 87 98 08 18 28 38 48 58 68 78 88 99 09 19 29 39 49 59 69 79 810 010 110 210 310 410 510 611 011 111 211 311 411 511 612 012 112 212 312 413 013 113 213 314 014 114 215 0

13.775.995.892.201.906.419.792.813.141.6410.439.622.525.241.663.184.967.401.883.943.680.426.695.102.637.705.945.576.004.294.409.294.816.534.147.344.861.972.824.432.506.678.892.126.572.715.424.705.875.631.354.651.575.452.182.031.762.053.905.236.491.993.015.903.186.03

13.175.463.514.042.404.698.192.253.962.859.488.842.945.022.353.394.787.562.674.063.552.116.045.603.587.594.264.615.454.854.327.944.785.982.695.983.412.643.443.623.545.517.662.665.833.125.072.923.815.572.586.232.825.202.372.542.832.183.104.385.703.252.725.973.385.07

0.360.100.881.370.080.430.270.320.160.280.050.460.090.490.080.280.640.400.230.090.250.050.180.450.320.300.780.210.450.100.140.210.400.200.150.130.440.620.190.250.270.280.250.320.390.360.160.220.510.170.120.330.450.330.090.120.210.260.160.740.840.090.200.240.262.46

13.555.735.003.011.815.339.032.492.621.419.439.913.474.601.772.453.957.491.793.533.571.656.085.523.147.963.964.034.435.273.947.575.006.613.445.793.881.413.763.032.895.947.602.045.732.165.174.054.335.791.514.321.756.013.162.032.252.073.405.265.362.683.284.943.105.49

0.190.350.020.160.130.200.190.140.180.050.270.320.190.030.230.070.170.220.230.160.180.080.120.180.170.120.270.140.160.020.280.080.100.080.190.260.090.110.230.150.200.170.140.090.210.020.350.320.120.090.140.200.220.170.480.140.230.130.020.250.250.160.370.200.160.16

* Projection structure factors for PM from Henderson et al. (1990).4 Each FN and FD are obtained through merge of, on average, 21.5 and 26.5 separate observations, respectively, for the same reflection and aregiven as,

N F./ N 1.0NE=N10-j=1 l/ j-=1 i

where oj is the standard deviation in the extracted sixfold averaged structure factor Fj for the jth pattern and N is the number of patterns averaged. Theoverall standard deviation Ur for the merged F values are given as

N = [/N_-F2 1.or EJ 2 N1072

j=1 ai ~~j=1 a

Volume 65 September 1993

2.25

D1.75> 1.50

0.75

0.50

0.25

0.000.00 0.05 0.10 0.15 1 0.20 0.25 0.30

RESOLUTION IN A

Electron Diffraction Studies on Expressed bR

2.50

2.25

2.00

1.75

Volume 65 September 1993

FIGURE 7 A "noise" map at 3.6-A resolution synthesized with ampli-tudes as the expected errors in the difference amplitudes FD - FN (seesection on Results for the method of calculating expected errors). The con-tour interval is the same as in Fig. 6 a.

bFIGURE 6 (a) Projection Fourier difference map calculated to 3.6-Aresolution using the difference amplitudes FD - FN I. Solid linesrepresent positive and dashed lines negative contour levels. The con-tour interval is one standard deviation fl of the expected noise level (Eq.2). (b) The difference map in a with the significant peaks shaded dark isoverlaid on the e-bR projection map. The locations in projection of the bRhelices A (amino-terminal) to G (carboxyl-terminal) and the position ofAsp96 as in the three-dimensional model of bR (Henderson et al., 1990) areindicated.

In the map shown in Fig. 6 a, the integrated scatteringpotentials of all peaks or valleys within and in the neigh-borhood of the protein envelope are

Electron Diffraction Studies on Expressed bR

extra amino-terminal residues in the fusion protein. The in-tegrated potential of 7 C-atoms in this peak corresponds toonly about 1.5 amino acids, far less than the expected -10amino acids based on the estimated proportions of the amino-terminal-processed bR polypeptides in PM (Miercke et al.,1989) and in e-bR (Shand et al., 1991). Even considering thatany signal in a difference-Fourier map is expected to appearat half its actual size, this peak would correspond at best tothree amino acids or to about 30% occupancy of the extraamino terminus amino acids in the fusion protein. This wouldsuggest that the elongated amino terminus in this bR analogis quite disordered as in PM (Henderson et al., 1990).

e-D96N

It is known that azide, sometimes used as an antimicrobialagent in buffer solutions, can functionally replace D96 inD96N as a surrogate proton donor and acceptor (Tittor et al.,1989) to reversibly restore wild-type activity. To separate anypossible electronic and/or induced structural effect of azideon the mutant protein structure, we removed azide from crys-tallized e-D96N prior to sample preparation for electron dif-fraction by washing with large excess of azide-free buffer.

Table 3 shows that the estimated "true" averaged fractionalchange in intensities between e-D96N and e-bR data definedas the difference of the observed and the estimated contri-bution of noise is largest (9.8%) at high resolution (5-3.6 A)with small contributions (-3.3%) at low resolution. Thismeans that relative to e-bR changes in e-D96N primarilyinvolve movements of atoms through distances that are likelyto be -4 A or less. The "noise" map shown in Fig. 7 syn-thesized with the estimated errors in the e-D96N-e-bR dif-ference amplitudes describes the sizes of peaks and valleysthat are expected from the contribution ofrandom noise alonein the consensus difference map shown in Fig. 6 a. The mini-mum and maximum values of density in the "noise" map wasfound to be very close to -3fQ and 3fQ, respectively (fl is theestimated standard deviation in the noise level of the dif-ference map in Fig. 6 a calculated using Eq. 2) which is equalto the threshold at which a peak or a valley is consideredstatistically reliable in a map.The magnitudes of the changes between e-D96N and e-bR

given as the integrated densities of the peaks and valleys inthe difference map (Fig. 6 a) are small being

1304 Biophysical Journal Volume 65 September 1993

FIGURE 8 Digitized display of high resolutionelectron diffraction pattern from glucose embed-ded h-D96N crystal after subtracting contributionfrom inelastic scatter. The two major overlappinglattices are inclined to each other by an angle of40. Diffraction spots to 2.9 A are visible. Thecircular arcs represent 3.1-A resolution.

We have in this work established a formalism for inves-tigating induced structural changes at high resolution at thelevel of 2-3 carbon atom equivalents in bacteriorhodopsinvariants. With the advent of aDNAtransformation system forHalobacterium (Cline et al., 1989) such investigations on H.Halobium-expressed bR variants that form highly orderedcrystalline arrays in vivo (Fig. 8) are possible for the groundstate and in trapped intermediates.

CONCLUSION

1) We have developed a protocol that generates large(-5-,um average diameter) 2-D crystals of E. coli-expressedbacteriorhodopsin and its variant D96N starting with opsinprotein purified in SDS. These crystals diffract to better than3.0-A resolution at room temperature and have a lattice ho-mologous to the P3 lattice in PM or in transformed PM. Thisis the first instance of an integral membrane protein yielding2-D crystals of such high order after being purified from thedenaturing detergent SDS.

2) The structural alterations determined in E. coli-expressed variants should faithfully reproduce the situationin the PM. The Fourier difference map at 3.6-A resolution(Fig. 4 b) shows that the structure of e-bR is similar to thatof w-bR in PM with no global structural rearrangements.This agrees with our previous similar conclusions from ob-

servations that e-bR has a rate of retinal incorporation, darkand light-adapted absorption properties, light-induced protonpumping activity (Miercke et al., 1991), photocycle kinetics(Milder et al., 1991), and chromophore geometry and envi-ronment (Lin et al., 1991) identical to those for w-bR.

3) The difference map (Fig. 6 a) shows that for the isos-teric substitution of Asp96 by Asn, the molecular conforma-tion in the fusion bR analog in the ground state is essentiallyunaltered. This, therefore, proves that the known effect of thismutation in terms of slowing down the decay of the M412intermediate (Holz et al., 1989) is not due to ground-statestructural perturbations and that the carboxylate moiety isindeed critical for the proton translocation.

This work was supported by the National Institutes of Health grantsGM32079 (to R. M. Stroud) and GM31785 (to M. C. Betlach).

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