JOURNAL OF ULTRASTRUCTURE RESEARCH 66, 235-242 (1979)
Structure of the Surface Layer Protein of the Outer Membrane of
Spirillum serpens R. M. GLAESER, WAHCHIU, AND DAVID GRANO Division of Medical Physics and Donner Laboratory, University of California, Berkeley, CA 94720 Received April 14, 1978
The outer membrane of the Gram negative bacterium, Spirillum serpens VHA, possesses an ordered surface-layer protein. A morphological model of this protein is proposed on the basis of electron micrographs that have been obtained of unstained, hydrated specimens as well as of negatively stained specimens. The molecular weight of the protein monomer in this model is consistent with the surface-layer protein molecular weight obtained by gel electrophoresis and estimated to be 140,000. In addition, gel electrophoresis reveals the presence of proteins of MW -~35,000 and MW -78,000, which remain associated with the outer membrane under conditions where the ordered surface-layer protein is released in soluble form. It has been known for some time, from the work of M u r r a y (1963) and of Buckmire and M u r r a y (1970, 1973, 1976), t h a t the outer m e m b r a n e of the G r a m negative bact e r i u m S p i r i l l u m serpens V H A possesses a surface-layer protein, which exhibits a regular hexagonal packing of the morphological sub-units. T h e en face view of the hexagonally packed (HP) protein layer shows globular subunits, which have an a p p a r e n t hole in their center and which are attached to one a n o t h e r by a relatively faint, threepronged connection (referred to in the literature as a "Y-linker" structure). T h e HPprotein is a t t a c h e d to the outer surface of the outer m e m b r a n e of this G r a m negative bacterium. T h e material to which the HPprotein is a t t a c h e d has been referred to as a "backing layer" in the studies of M u r r a y and co-workers. T h e H P - p r o t e i n can be released from the outer m e m b r a n e by mild guanidine hydrochloride t r e a t m e n t (Buckmire and Murray, 1970, 1973). T h e HP-protein attaches again to the outer membrane, in an ordered structure, on removal of the guanidine hydrochloride by dialysis and on addition of calcium ion (Buckmire and Murray, 1973, 1976). T h e surface-layer protein of S p i r i l l u m serpens a p p a r e n t l y will not form a two-dimensional array in the absence of outer m e m b r a n e . In some other
organisms the surface protein can be reconstituted to form a two-dimensional lattice b o t h in the presence and in the absence of a natural substrate (Aebi et al., 1973; Sleytr, 1975; T h o r n l e y et al., 1974). Our attention has been drawn to the HPprotein layer of S p i r i l l u m serpens as possibly being suitable for three-dimensional structure analysis by electron microscopy. This structure also offers the possibility of providing crystallographic information on the detailed association of a surface protein with the underlying lipopolysaccharide a n d / o r other components of the outer bacterial membrane. Of course, this information will be available only if a high resolution structure analysis proves to be possible. Considerable biochemical analysis has already been performed on the surfacelayer protein as well as the whole cell-wall material of S p i r i l l u m serpens. T h e HP-protein appears to have a m o n o m e r molecular weight of about 140,000 to 150,000 daltons, as d e t e r m i n e d by SDS gel electrophoresis (Buckmire and Murray, 1973). On the other h a n d b o t h the sedimentation velocity of H P - p r o t e i n in sucrose gradients, in the presence of guanidine hydrochloride, and the amino acid composition would be consistent with a m o n o m e r molecular weight
235 0022-5320/79/030235-08502.00/0 Copyright© 1979by AcademicPress, Inc. All rights of reproductionin any formreserved.
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of ~50,000 (Buckmire a n d M u r r a y , 1973). T h e lipid analysis of whole cell-walls h a s b e e n r e p o r t e d (Chester a n d M u r r a y , 1975), considerable a t t e n t i o n having b e e n given to the lipopolysaccharide c o m p o n e n t . A morphological m o d e l of the s t r u c t u r e of t h e H P - p r o t e i n h a s b e e n p r o p o s e d b y B u c k m i r e a n d M u r r a y (1976). T h i s m o d e l a s s u m e s t h a t the m o n o m e r m o l e c u l a r weight of the protein is ~48,000, a n d t h a t these m o n o m e r s are associated in t r i m e r s having a three-fold, spoke-like structure. T h e t r i m e r s in t u r n are associated with one a n o t h e r as h e x a m e r s to give a morphological unit consisting of a large globular struct u r e with a central hole and six radial spokes, which constitute the Y-linkers seen in the intact structure. A special feature of this m o d e l is t h a t t h r e e of the radial spokes or Y-linkers lie at one level in the s t r u c t u r e a n d t h r e e lie at a second level. W e wish to p r o p o s e h e r e an alternative morphological m o d e l in which the morphological unit m i g h t be described as having the appearance of a flared-out, hollow cylinder with six " s p o k e s " at the flared end (refer to the discussion section for a sketch of this model). T h i s alternative m o d e l h a s b e e n suggested b y images o b t a i n e d f r o m outer cell-wall m a t e r i a l p r e p a r e d in an unfixed, unstained, frozen h y d r a t e d state. T h e m o d e l h a s b e e n f u r t h e r confirmed b y side views of t h e H P - p r o t e i n layer a t t a c h e d to the o u t e r m e m b r a n e , as seen in negatively stained specimens. Finally, the m o d e l is consistent with the strong evidence provided b y S D S gel electrophoresis, t h a t the m o n o m e r m o l e c u l a r weight of the protein is close to 140,000 r a t h e r t h a n being close to 50,000. MATERIALS AND METHODS The outer cell wall material of Spirillum serpens has been isolated by the method of Buckmire and Murray (1970). This method relies on the fact that the HP-protein, attached to the outer membrane, is sloughed off from intact bacteria by brief treatment at 60°. The outer surface layer material is then collected and washed by centrifugation. Material isolated in this way consists of flat patches of variable size, in which
the protein shows hexagonal packing. In addition a variety of tubular forms are seen, as well as areas of outer membrane material on which no surface protein can be seen. This isolated outer membrane material was used for electron microscopy "as is," without further attempts at fractionation or purification. Specimens were prepared for electron microscopy by negative staining with 1 per cent potassium phosphotungstate at pH 7.0. In addition, unstained, frozen hydrated specimens were prepared as described by Taylor and Glaeser {1976). Computer processing of electron micrographs was carried out with programs developed for the CDC 7600 system at the Lawrence Berkeley Laboratory (Grano, 1979). Photographic plates were digitized with the PDS scanning densitometer located in the Astronomy Department, University of California, Berkeley. Image interpolation and processing were carried out by procedures similar to those described by Aebi et al. (1973). Sohm features of the image processing programs of DeRosier and Moore (1970) were also used for the analysis of tubular forms. Polyacryl~amide gel electrophoresis in sodium dodecyl sulfate (SDS) was performed as described by Fairbanks et-al. (1971}, using the sample buffer of Laemmli (1970). The sample buffer contains 0.0625 M tris (hydroxymethyl) aminomethane (tris) hydrochloride (pH 6.8), 2 per cent SDS, 5 per cent mercaptoethanol, 10 per cent glycerol and 0.001 per cent bromophenol blue. The protein standards for molecular weight estimates were RNA polymerase (/T chain -160,000 and fl chain = 150,000) kindly provided by Dr. Leroy Liu, bovine serum albumin (68,000), ovalbumin (45,000), gp32 protein of T-4 bacteriophage {35,000) kindly provided by Dr. Junko H0soda, and chymotrypsinogen A (25,000). All samples were solubilized in the sample buffer by heating at 100° for 10 minutes. RESULTS Face-on views of flat patches, in which the hexagonal packing of H P - p r o t e i n s could be seen, showed the s a m e morphological details t h a t h a v e already b e e n described in the literature. T h i s s t a t e m e n t applies to the frozen h y d r a t e d specimens as well as to t h e negatively stained specimens, a l t h o u g h it m u s t be m e n t i o n e d t h a t images of t h e unstained, h y d r a t e d s p e c i m e n show reverse c o n t r a s t in c o m p a r i s o n to t h a t of the negatively stained specimens ( c o m p a r e Figure l a and lb). I n images of frozen h y d r a t e d , u n s t a i n e d biological m a t e r i a l s the p r o t e i n is expected to be d a r k c o m p a r e d to t h e surrounding w a t e r (i.e. ice), since the m a s s
SURFACE LAYER PROTEIN density of protein is 1.3 to 1.5 times the density of water. The connection between morphological subunits (i.e., the Y-linker structure) is difficult to see in the original micrographs of unstained, hydrated HP-protein layer. However, computer averaging of the unstained specimens out to a resolution of 22 ,h, results in the image shown in Figure lc, which clearly demonstrates that the bridging structure between morphological units is present in the images of unstained, hydrated material. The face-on view shows
237
the main portion of the HP-protein to have a diameter of -85/~, an inner hole of ~25 /~ diameter, and a center-to-center distance of 145/~. The accuracy of these measurements is, of course, limited by the moderate resolution at this stage of the structure analysis. The frozen specimen preparation furthermore gives some images that have no counterpart in the negatively stained material. The unit membrane appearance of the unfixed, unstained outer membrane has already been noted by Taylor, Grano and
FIG. 1. Face-onviews of the hexagonallypacked protein of S. serpens, attached to fragmentsof the outer membrane: a) negativelystained with potasium phosphotungstate,b) unstained,frozenhydrated specimen,c) spatially averagedimage derivedfrom the well orderedportion of (b) by computerprocessing;protein is shown darker than the backgroundin the computer display.The bar indicates500 A.
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GLAESER, CHIU, AND GRANO
Chiu (1976) and by Taylor (1978). In addition, a variety of side views of the HPprotein, still attached to the unit membrane, can be seen in the frozen hydrated specimen. These side views are valuable in that they give a relatively accurate value for the thickness (~ 155A) of the HP -protein layer. The side views further show that the region of greatest protein density, seen side on, occurs at the outer surface of the HPprotein layer. This observation suggests that the "Y-linker region," that is the region of protein contacts, occurs only at the outer aqueous surface. This conflicts with the model of Buckmire and Murray (1976) in which protein-protein contacts must occur at two levels. Among the variety of side views obtained with the frozen hydrated specimens there is a unique view already published in the paper of Taylor (1977), which we reproduce here at higher magnification in Figure 2a. We now suggest that this unique view is possibly an image of just two HP-protein morphological units, attached to a minute, spherical vesicle of the outer bacterial membrane, and in contact with one another. This particular view served as the first suggestion for what we now propose as
a low-resolution model of the HP-protein structure. We interpret the image in Figure 2a to imply that the morphological unit is cylindrical in shape but flared out towards the outer surface. With this suggestion in mind, we have examined a large number of negatively stained specimens, and they too show side views that are consistent with the model described here and in the discussion. An example of a corroborating image obtained from negatively stained specimens is shown in Figure 2b. Finally, we had hoped to get further information on the low resolution structure of the HP-protein by the use of three-dimensional, helical reconstruction applied to images of the tubular forms that are always present as a result of the isolation procedure. Our efforts in this direction have been frustrated by the fact that all the tubular forms analyzed thus far have had cylindrical symmetry rather than true helical symmetry. Optical diffraction patterns from several tubes can be fit by the selection rule 1 = 2n + m (Mellema and van den Berg, 1974), and the diffraction maxima can be overlayed by a hexagonal net. The layerline spacing of 1/145A -1 is consistent with the center-to-center distance of the sub-
FIG. 2. Side views of the HP protein of S. serpens outer membrane: a) an unstained, frozen hydrated specimen showing (apparently) only two morphologicalunits attached to a small, spherical vesicle of outer membranematerial, b) a longtubular portion of outer membranematerial, on whichnumerousHP units can be seen in a clear side view.The bar indicates500 A.
239
SURFACE LAYER PROTEIN
units in the projected planar views of HPprotein. This evidence leads to a view of the tubes as structures derived from rolledup sheets. The possibility of a hexagonal plane lattice undergoing such a transformation is discussed by Kiselev and Klug (1969). A detailed analysis of the tubes has shown that the phase relationships in the computed Fourier transforms of the tubes in consistent with cylindrical symmetry (Grano, 1979). As previously explained by Mellema and van den Berg, cylindrical symmetry causes problems with optical filtration and 3-D reconstruction attempts. Each layer-line contains contributions from Bessel functions of both positive and negative order n', where n' is allowed by the selection rule. The interference between these Bessel functions varies with the azimuthal angle. The method of helical reconstruction based on a single projection of the helix (De Rosier & Klug, 1968) depends on there being a single Bessel-function contribution in the near axial portion of each layer line. In order to process the interfering Bessel functions, at least two projected views of known relative orientation would be needed. At the present time computer programs to accomplish this separation. have not been implemented. Polyacrylamide gel electrophoresis of the isolated outer membrane material reveals a band at molecular weight ~140,000 (the H P protein), a pair of bands at ~78,000, and a pair of bands at ~35,000, as shown in Figure 3b. Using an approach similar to that of Buckmire and Murray, the periodic H P protein was released in soluble form by treating the outer membrane material in 2.0 M guanidine hydrochloride for 4 hours at 20 °. The solubilized H P protein was then separated from the insoluble components by centrifugation. Figures 3a and 3c show the gel electrophoresis patterns of the soluble and insoluble fractions, respectively. If the SDS gels are stained for carbohydrate by the Periodic Acid-Schiff (PAS) procedure before being stained with coomassie
brilliant blue for protein, only the band at 140,000 shows a faint color. This may mean that the H P protein is a glycoprotein, and in addition the small amount of carbohydrate present may cause the estimated molecular weight value to be too large. The membrane proteins of molecular weight ~35,000 and ~78,000 that we have observed here have not been reported previously for S. serpens, outer membrane, A
B
C
FIG. 3. Gel electrophoresis patterns produced by the outer membrane material of S. serpens after staining with coomassie brilliant blue: a) the isolated HP protein material that is released from the outer membrane preparation by 2.0 M guanidine hydrochloride, b) the whole outer membrane preparation, from which the HP-protein in (a) was derived, c) the guanidine hydrochloride insoluble fraction produced during the preparation of (a).
240
GLAESER, CHIU, AND GRANO
although proteins of similar molecular weight have been reported for the outer membranes of the Gram negative bacteria E. coli (Rosenbusch, 1974) and Salmonella typhimurium (Ames, et al., 1974). The solubilization of the 35,000 MW protein component in SDS buffer requires heating at 60 ° for half an hour or at 100 ° for at least half a minute. Buckmire and Murray (1973) noted that protein material was present in the guanidine hydrochloride insoluble fraction, but they did not report any attempt to analyze that material by gel electrophoresis. We note, finally, that the strong solubilization conditions used in the present work still result in the H P protein appearing as a single band of M W -140,000. DISCUSSION
The model proposed here for the morphological appearance of the hexagonally packed protein in SpiriUum serpens is illustrated in Figure 4. Figure 4a shows a
perspective view representing the morphological units packed in an hexagonal array on the surface of the outer membrane. A single morphological unit is shown in a highly schematic, exploded view in Figure 4b. As shown in these figures, we believe that the morphological unit is a hexamer of proteins that have a monomer molecular weight of 140,000. We have no information at the present time as to the detailed shape of the protein monomer, and the "exploded" sub unit shown in Figure 4b should not be regarded as having the actual structure of the monomer unit. The structure of the hexamer is represented in Figure 4 as a cylindrical tube, of outer diameter approximately 85/~, with an inner pore of diameter approximately 25/~. The base of the cylinder is attached to the surface of the outer bacterial membrane. The top of the cylinder is split, and it flares open to form a six fold "spoke" or umbrella-type structure, the tips of which form the points of contact
?
c) ~i~6o-7oA
:~
b)
~
Ii i I I! ii
d)
FIG. 4. The model proposed for the low resolution structure of the HP protein ofS. serpens. Figure 4a shows a perspective view of the proposed packing and protein-to-protein contacts between individual morphological units. Figure 4b shows a blow-up of a single morphological unit. The morphology of the exploded subunit is not meant to suggest the actual shape of individual protein monomers, as no experimental information is yet available on the shapes or contours of the protein within the main, cylindrical portion of the structure.
241
SURFACE LAYER PROTEIN
between adjacent morphological units. It is to be emphasized that we have no information yet on the shape of the proteinprotein contacts within the region of the main body of the cylinder. Therefore no imagined protein shape in this region is indicated. The suggestion that the morphological unit of the HP-protein is in fact a hexamer of proteins with molecular weight of 140,000 is very consistent with the measured size of the morphological unit. The estimated volume of the morphological unit illustrated in Figure 4 is approximately 10.4 × 105 /~3. This estimate represents the volume of an 85 /~ diameter cylinder that is 155/~ tall with a hollow center 25/~ in diameter, plus the volume of six cylinders (the "Y-linders") that are 25 A in diameter and 80/~ long. The volume of the protein monomer (i.e. one sixth of the morphological unit) would then be about 1.7 × 105 A 3. The molecular weight of the monomer would be about 150,000, if we assume a mass density of 1.5 grams/cm 3 (i.e. the dry density of protein) or about 130,000 if we assume a mass density of 1.3 grams/cm 3 (i.e. the buoyant density of many hydrated protein materials). Taking into consideration the inaccuracy of measuring the dimensions quoted above, these values of the calculated molecular weight are entirely consistent with the experimentally estimated value of 140,000. Buckmire and Murray have given some biochemical evidence that would be consistent with a lower monomer molecular weight (Buckmire and Murray, 1973, 1976). We believe, however, that the reliability of the SDS polyacrylamide gel electrophoresis method of determining molecular weights is great enough that the value MW -140,000 should serve as a strong constraint on the validity of any model. The particularly vigorous conditions of protein solubilization in SDS that we have used give further confidence that the HP protein band at MW ~ 140,000 does not represent a dimer (or higher aggregate) of peptides with
lower molecular weights. More detailed information regarding the structure of the HP-protein, and the structural relationships involved in its association with the material of the outer bacterial membrane, requires first of all that images of unfixed, unstained protein be obtained at lower electron exposures than those obtained in the present work. The resulting low-dose images should be superimposed and averaged to obtain high resolution data (Kuo and Glaeser, 1975; Unwin and Henderson, 1975). Work of this type can be carried out by developing appropriate cross correlation methods, similar to those described by Saxton and Frank (1977), to superimpose a large number of patches of hexagonally packed material, since the individual patches have too few repeating units to provide the needed high resolution data. Alternatively the procedure for isolating the HP-protein layer may be susceptible to modifications, which would yield larger, single patches with good crystalline order. Finally, it will be necessary to collect image data with tilted specimens and to retrieve the full, three-dimensional structure from a large number of different, tilted views. We wish to thank Prof. R. G. Murray for supplying us with the initial culture of Spirillum serpens strain VHA. We also t h a n k Dr. Ivy Kuo, who conducted preliminary studies in our laboratory; Mrs. Thea Scott-Garner, who assisted with the isolation of the H P layer and with the gel electrophoresis studies; and Dr. Kenneth Taylor for the use of the micrograph from which Figure 2a was extracted. This research was supported by the Department of Energy contract # W-7405-ENG-48 and NIH Grant # G M 23325. REFERENCES
1. AEBI, U., SMITH, P. R., DUBOCHET,J., HENRY, C., AND KELLENBERGER, E. (1973) J. Supramol. Struct. 1, 498-522. 2. AMES, G. F. L., SPUDICH, E. N., AND NIKAIDO, H. (1974) J. Bacteriol. 117, 406-416. 3. BUCKMIRE, F. L. A., AND MURRAY, R. G. E. (1970) Can. J. Microbiol. 16, 1011-1022. 4. BURKMIRE, F. L. A., AND MURRAY, R. G. E. (1973) Can. J. Mierobiol. 19, 59-66. 5. BUCKMIRE,F. L. A., AND MURRAY,R. G. E. (1976) J. Bacteriol. 125, 290-299.
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6. CHESTER, I. R., AND MURRAY, R. G. E (1975) J. Bacteriol. 124, 1168-1176. 7. DERoSIER, D. J. ANDMOORE, P. B., J. Mol. Biol. 52, 355 (1970). 8. FAIl'BANKS, G., STECK, T. L., AND WALLACH, D. F. H. (1971) Biochem. 10, 2606-2617. 9. GRANO, D. A. (1979) Ph.D. Thesis, University of California, Berkeley. 10. KISELEV, N. A. AND KLUG, A. (1969) J. Mol. Biol. 40, 155-171. 11. Kuo, I. A. M., AND GLAESER, R.M. (1975) Ultramicroscopy 1, 53-66. 12. LAEMMLI,U. K. (1970) Nature 227, 680-685. 13. MELLEMA, J. E., AND VAN DEN BERG, H. J. N. (1974) J. Supramol. Struct. 2, 17-31. 14. MURRAY, R. G. E. (1963) Can. J. Microbiol. 9, 381-392.
15. ROSENBUSCH, J. P. (1974) J. Biol. Chem. 249, 8019-8029. 16. SAXTON, W. 0., AND FRANK, J. (1977) Ultramicroscopy 2, 219-227. 17. SLEYTR, U. B. (1976) J. Ultrastruct. Res. 55, 360377. 18. TAYLOR,K. A. (1978) J. Microscopy 112, 115-125. 19. TAYLOR, K. A., AND GLAESER, R. M. (1976) J. Ultrastruct. Res. 55, 448--456. 20. TAYLOR, K. A., GRANO, D. A., AND CHIU, W. (1976) Proceedings 34th Annual Meeting EMSA, 136-137. 21. THORNLEY, M. J., GLAUERT,A. M., AND SLEYTR, U. B. (1974) Phil. Trans. R. Soc. Lond. B. 268, 147-153. 22. UNWIN, P. N. T., AND HENDERSON, R. (1975) J. Mol. Biol. 94, 425-440.
Structure of the Surface Layer Protein of the Outer Membrane of
Spirillum serpens R. M. GLAESER, WAHCHIU, AND DAVID GRANO Division of Medical Physics and Donner Laboratory, University of California, Berkeley, CA 94720 Received April 14, 1978
The outer membrane of the Gram negative bacterium, Spirillum serpens VHA, possesses an ordered surface-layer protein. A morphological model of this protein is proposed on the basis of electron micrographs that have been obtained of unstained, hydrated specimens as well as of negatively stained specimens. The molecular weight of the protein monomer in this model is consistent with the surface-layer protein molecular weight obtained by gel electrophoresis and estimated to be 140,000. In addition, gel electrophoresis reveals the presence of proteins of MW -~35,000 and MW -78,000, which remain associated with the outer membrane under conditions where the ordered surface-layer protein is released in soluble form. It has been known for some time, from the work of M u r r a y (1963) and of Buckmire and M u r r a y (1970, 1973, 1976), t h a t the outer m e m b r a n e of the G r a m negative bact e r i u m S p i r i l l u m serpens V H A possesses a surface-layer protein, which exhibits a regular hexagonal packing of the morphological sub-units. T h e en face view of the hexagonally packed (HP) protein layer shows globular subunits, which have an a p p a r e n t hole in their center and which are attached to one a n o t h e r by a relatively faint, threepronged connection (referred to in the literature as a "Y-linker" structure). T h e HPprotein is a t t a c h e d to the outer surface of the outer m e m b r a n e of this G r a m negative bacterium. T h e material to which the HPprotein is a t t a c h e d has been referred to as a "backing layer" in the studies of M u r r a y and co-workers. T h e H P - p r o t e i n can be released from the outer m e m b r a n e by mild guanidine hydrochloride t r e a t m e n t (Buckmire and Murray, 1970, 1973). T h e HP-protein attaches again to the outer membrane, in an ordered structure, on removal of the guanidine hydrochloride by dialysis and on addition of calcium ion (Buckmire and Murray, 1973, 1976). T h e surface-layer protein of S p i r i l l u m serpens a p p a r e n t l y will not form a two-dimensional array in the absence of outer m e m b r a n e . In some other
organisms the surface protein can be reconstituted to form a two-dimensional lattice b o t h in the presence and in the absence of a natural substrate (Aebi et al., 1973; Sleytr, 1975; T h o r n l e y et al., 1974). Our attention has been drawn to the HPprotein layer of S p i r i l l u m serpens as possibly being suitable for three-dimensional structure analysis by electron microscopy. This structure also offers the possibility of providing crystallographic information on the detailed association of a surface protein with the underlying lipopolysaccharide a n d / o r other components of the outer bacterial membrane. Of course, this information will be available only if a high resolution structure analysis proves to be possible. Considerable biochemical analysis has already been performed on the surfacelayer protein as well as the whole cell-wall material of S p i r i l l u m serpens. T h e HP-protein appears to have a m o n o m e r molecular weight of about 140,000 to 150,000 daltons, as d e t e r m i n e d by SDS gel electrophoresis (Buckmire and Murray, 1973). On the other h a n d b o t h the sedimentation velocity of H P - p r o t e i n in sucrose gradients, in the presence of guanidine hydrochloride, and the amino acid composition would be consistent with a m o n o m e r molecular weight
235 0022-5320/79/030235-08502.00/0 Copyright© 1979by AcademicPress, Inc. All rights of reproductionin any formreserved.
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GLAESER, CHIU, AND GRANO
of ~50,000 (Buckmire a n d M u r r a y , 1973). T h e lipid analysis of whole cell-walls h a s b e e n r e p o r t e d (Chester a n d M u r r a y , 1975), considerable a t t e n t i o n having b e e n given to the lipopolysaccharide c o m p o n e n t . A morphological m o d e l of the s t r u c t u r e of t h e H P - p r o t e i n h a s b e e n p r o p o s e d b y B u c k m i r e a n d M u r r a y (1976). T h i s m o d e l a s s u m e s t h a t the m o n o m e r m o l e c u l a r weight of the protein is ~48,000, a n d t h a t these m o n o m e r s are associated in t r i m e r s having a three-fold, spoke-like structure. T h e t r i m e r s in t u r n are associated with one a n o t h e r as h e x a m e r s to give a morphological unit consisting of a large globular struct u r e with a central hole and six radial spokes, which constitute the Y-linkers seen in the intact structure. A special feature of this m o d e l is t h a t t h r e e of the radial spokes or Y-linkers lie at one level in the s t r u c t u r e a n d t h r e e lie at a second level. W e wish to p r o p o s e h e r e an alternative morphological m o d e l in which the morphological unit m i g h t be described as having the appearance of a flared-out, hollow cylinder with six " s p o k e s " at the flared end (refer to the discussion section for a sketch of this model). T h i s alternative m o d e l h a s b e e n suggested b y images o b t a i n e d f r o m outer cell-wall m a t e r i a l p r e p a r e d in an unfixed, unstained, frozen h y d r a t e d state. T h e m o d e l h a s b e e n f u r t h e r confirmed b y side views of t h e H P - p r o t e i n layer a t t a c h e d to the o u t e r m e m b r a n e , as seen in negatively stained specimens. Finally, the m o d e l is consistent with the strong evidence provided b y S D S gel electrophoresis, t h a t the m o n o m e r m o l e c u l a r weight of the protein is close to 140,000 r a t h e r t h a n being close to 50,000. MATERIALS AND METHODS The outer cell wall material of Spirillum serpens has been isolated by the method of Buckmire and Murray (1970). This method relies on the fact that the HP-protein, attached to the outer membrane, is sloughed off from intact bacteria by brief treatment at 60°. The outer surface layer material is then collected and washed by centrifugation. Material isolated in this way consists of flat patches of variable size, in which
the protein shows hexagonal packing. In addition a variety of tubular forms are seen, as well as areas of outer membrane material on which no surface protein can be seen. This isolated outer membrane material was used for electron microscopy "as is," without further attempts at fractionation or purification. Specimens were prepared for electron microscopy by negative staining with 1 per cent potassium phosphotungstate at pH 7.0. In addition, unstained, frozen hydrated specimens were prepared as described by Taylor and Glaeser {1976). Computer processing of electron micrographs was carried out with programs developed for the CDC 7600 system at the Lawrence Berkeley Laboratory (Grano, 1979). Photographic plates were digitized with the PDS scanning densitometer located in the Astronomy Department, University of California, Berkeley. Image interpolation and processing were carried out by procedures similar to those described by Aebi et al. (1973). Sohm features of the image processing programs of DeRosier and Moore (1970) were also used for the analysis of tubular forms. Polyacryl~amide gel electrophoresis in sodium dodecyl sulfate (SDS) was performed as described by Fairbanks et-al. (1971}, using the sample buffer of Laemmli (1970). The sample buffer contains 0.0625 M tris (hydroxymethyl) aminomethane (tris) hydrochloride (pH 6.8), 2 per cent SDS, 5 per cent mercaptoethanol, 10 per cent glycerol and 0.001 per cent bromophenol blue. The protein standards for molecular weight estimates were RNA polymerase (/T chain -160,000 and fl chain = 150,000) kindly provided by Dr. Leroy Liu, bovine serum albumin (68,000), ovalbumin (45,000), gp32 protein of T-4 bacteriophage {35,000) kindly provided by Dr. Junko H0soda, and chymotrypsinogen A (25,000). All samples were solubilized in the sample buffer by heating at 100° for 10 minutes. RESULTS Face-on views of flat patches, in which the hexagonal packing of H P - p r o t e i n s could be seen, showed the s a m e morphological details t h a t h a v e already b e e n described in the literature. T h i s s t a t e m e n t applies to the frozen h y d r a t e d specimens as well as to t h e negatively stained specimens, a l t h o u g h it m u s t be m e n t i o n e d t h a t images of t h e unstained, h y d r a t e d s p e c i m e n show reverse c o n t r a s t in c o m p a r i s o n to t h a t of the negatively stained specimens ( c o m p a r e Figure l a and lb). I n images of frozen h y d r a t e d , u n s t a i n e d biological m a t e r i a l s the p r o t e i n is expected to be d a r k c o m p a r e d to t h e surrounding w a t e r (i.e. ice), since the m a s s
SURFACE LAYER PROTEIN density of protein is 1.3 to 1.5 times the density of water. The connection between morphological subunits (i.e., the Y-linker structure) is difficult to see in the original micrographs of unstained, hydrated HP-protein layer. However, computer averaging of the unstained specimens out to a resolution of 22 ,h, results in the image shown in Figure lc, which clearly demonstrates that the bridging structure between morphological units is present in the images of unstained, hydrated material. The face-on view shows
237
the main portion of the HP-protein to have a diameter of -85/~, an inner hole of ~25 /~ diameter, and a center-to-center distance of 145/~. The accuracy of these measurements is, of course, limited by the moderate resolution at this stage of the structure analysis. The frozen specimen preparation furthermore gives some images that have no counterpart in the negatively stained material. The unit membrane appearance of the unfixed, unstained outer membrane has already been noted by Taylor, Grano and
FIG. 1. Face-onviews of the hexagonallypacked protein of S. serpens, attached to fragmentsof the outer membrane: a) negativelystained with potasium phosphotungstate,b) unstained,frozenhydrated specimen,c) spatially averagedimage derivedfrom the well orderedportion of (b) by computerprocessing;protein is shown darker than the backgroundin the computer display.The bar indicates500 A.
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GLAESER, CHIU, AND GRANO
Chiu (1976) and by Taylor (1978). In addition, a variety of side views of the HPprotein, still attached to the unit membrane, can be seen in the frozen hydrated specimen. These side views are valuable in that they give a relatively accurate value for the thickness (~ 155A) of the HP -protein layer. The side views further show that the region of greatest protein density, seen side on, occurs at the outer surface of the HPprotein layer. This observation suggests that the "Y-linker region," that is the region of protein contacts, occurs only at the outer aqueous surface. This conflicts with the model of Buckmire and Murray (1976) in which protein-protein contacts must occur at two levels. Among the variety of side views obtained with the frozen hydrated specimens there is a unique view already published in the paper of Taylor (1977), which we reproduce here at higher magnification in Figure 2a. We now suggest that this unique view is possibly an image of just two HP-protein morphological units, attached to a minute, spherical vesicle of the outer bacterial membrane, and in contact with one another. This particular view served as the first suggestion for what we now propose as
a low-resolution model of the HP-protein structure. We interpret the image in Figure 2a to imply that the morphological unit is cylindrical in shape but flared out towards the outer surface. With this suggestion in mind, we have examined a large number of negatively stained specimens, and they too show side views that are consistent with the model described here and in the discussion. An example of a corroborating image obtained from negatively stained specimens is shown in Figure 2b. Finally, we had hoped to get further information on the low resolution structure of the HP-protein by the use of three-dimensional, helical reconstruction applied to images of the tubular forms that are always present as a result of the isolation procedure. Our efforts in this direction have been frustrated by the fact that all the tubular forms analyzed thus far have had cylindrical symmetry rather than true helical symmetry. Optical diffraction patterns from several tubes can be fit by the selection rule 1 = 2n + m (Mellema and van den Berg, 1974), and the diffraction maxima can be overlayed by a hexagonal net. The layerline spacing of 1/145A -1 is consistent with the center-to-center distance of the sub-
FIG. 2. Side views of the HP protein of S. serpens outer membrane: a) an unstained, frozen hydrated specimen showing (apparently) only two morphologicalunits attached to a small, spherical vesicle of outer membranematerial, b) a longtubular portion of outer membranematerial, on whichnumerousHP units can be seen in a clear side view.The bar indicates500 A.
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SURFACE LAYER PROTEIN
units in the projected planar views of HPprotein. This evidence leads to a view of the tubes as structures derived from rolledup sheets. The possibility of a hexagonal plane lattice undergoing such a transformation is discussed by Kiselev and Klug (1969). A detailed analysis of the tubes has shown that the phase relationships in the computed Fourier transforms of the tubes in consistent with cylindrical symmetry (Grano, 1979). As previously explained by Mellema and van den Berg, cylindrical symmetry causes problems with optical filtration and 3-D reconstruction attempts. Each layer-line contains contributions from Bessel functions of both positive and negative order n', where n' is allowed by the selection rule. The interference between these Bessel functions varies with the azimuthal angle. The method of helical reconstruction based on a single projection of the helix (De Rosier & Klug, 1968) depends on there being a single Bessel-function contribution in the near axial portion of each layer line. In order to process the interfering Bessel functions, at least two projected views of known relative orientation would be needed. At the present time computer programs to accomplish this separation. have not been implemented. Polyacrylamide gel electrophoresis of the isolated outer membrane material reveals a band at molecular weight ~140,000 (the H P protein), a pair of bands at ~78,000, and a pair of bands at ~35,000, as shown in Figure 3b. Using an approach similar to that of Buckmire and Murray, the periodic H P protein was released in soluble form by treating the outer membrane material in 2.0 M guanidine hydrochloride for 4 hours at 20 °. The solubilized H P protein was then separated from the insoluble components by centrifugation. Figures 3a and 3c show the gel electrophoresis patterns of the soluble and insoluble fractions, respectively. If the SDS gels are stained for carbohydrate by the Periodic Acid-Schiff (PAS) procedure before being stained with coomassie
brilliant blue for protein, only the band at 140,000 shows a faint color. This may mean that the H P protein is a glycoprotein, and in addition the small amount of carbohydrate present may cause the estimated molecular weight value to be too large. The membrane proteins of molecular weight ~35,000 and ~78,000 that we have observed here have not been reported previously for S. serpens, outer membrane, A
B
C
FIG. 3. Gel electrophoresis patterns produced by the outer membrane material of S. serpens after staining with coomassie brilliant blue: a) the isolated HP protein material that is released from the outer membrane preparation by 2.0 M guanidine hydrochloride, b) the whole outer membrane preparation, from which the HP-protein in (a) was derived, c) the guanidine hydrochloride insoluble fraction produced during the preparation of (a).
240
GLAESER, CHIU, AND GRANO
although proteins of similar molecular weight have been reported for the outer membranes of the Gram negative bacteria E. coli (Rosenbusch, 1974) and Salmonella typhimurium (Ames, et al., 1974). The solubilization of the 35,000 MW protein component in SDS buffer requires heating at 60 ° for half an hour or at 100 ° for at least half a minute. Buckmire and Murray (1973) noted that protein material was present in the guanidine hydrochloride insoluble fraction, but they did not report any attempt to analyze that material by gel electrophoresis. We note, finally, that the strong solubilization conditions used in the present work still result in the H P protein appearing as a single band of M W -140,000. DISCUSSION
The model proposed here for the morphological appearance of the hexagonally packed protein in SpiriUum serpens is illustrated in Figure 4. Figure 4a shows a
perspective view representing the morphological units packed in an hexagonal array on the surface of the outer membrane. A single morphological unit is shown in a highly schematic, exploded view in Figure 4b. As shown in these figures, we believe that the morphological unit is a hexamer of proteins that have a monomer molecular weight of 140,000. We have no information at the present time as to the detailed shape of the protein monomer, and the "exploded" sub unit shown in Figure 4b should not be regarded as having the actual structure of the monomer unit. The structure of the hexamer is represented in Figure 4 as a cylindrical tube, of outer diameter approximately 85/~, with an inner pore of diameter approximately 25/~. The base of the cylinder is attached to the surface of the outer bacterial membrane. The top of the cylinder is split, and it flares open to form a six fold "spoke" or umbrella-type structure, the tips of which form the points of contact
?
c) ~i~6o-7oA
:~
b)
~
Ii i I I! ii
d)
FIG. 4. The model proposed for the low resolution structure of the HP protein ofS. serpens. Figure 4a shows a perspective view of the proposed packing and protein-to-protein contacts between individual morphological units. Figure 4b shows a blow-up of a single morphological unit. The morphology of the exploded subunit is not meant to suggest the actual shape of individual protein monomers, as no experimental information is yet available on the shapes or contours of the protein within the main, cylindrical portion of the structure.
241
SURFACE LAYER PROTEIN
between adjacent morphological units. It is to be emphasized that we have no information yet on the shape of the proteinprotein contacts within the region of the main body of the cylinder. Therefore no imagined protein shape in this region is indicated. The suggestion that the morphological unit of the HP-protein is in fact a hexamer of proteins with molecular weight of 140,000 is very consistent with the measured size of the morphological unit. The estimated volume of the morphological unit illustrated in Figure 4 is approximately 10.4 × 105 /~3. This estimate represents the volume of an 85 /~ diameter cylinder that is 155/~ tall with a hollow center 25/~ in diameter, plus the volume of six cylinders (the "Y-linders") that are 25 A in diameter and 80/~ long. The volume of the protein monomer (i.e. one sixth of the morphological unit) would then be about 1.7 × 105 A 3. The molecular weight of the monomer would be about 150,000, if we assume a mass density of 1.5 grams/cm 3 (i.e. the dry density of protein) or about 130,000 if we assume a mass density of 1.3 grams/cm 3 (i.e. the buoyant density of many hydrated protein materials). Taking into consideration the inaccuracy of measuring the dimensions quoted above, these values of the calculated molecular weight are entirely consistent with the experimentally estimated value of 140,000. Buckmire and Murray have given some biochemical evidence that would be consistent with a lower monomer molecular weight (Buckmire and Murray, 1973, 1976). We believe, however, that the reliability of the SDS polyacrylamide gel electrophoresis method of determining molecular weights is great enough that the value MW -140,000 should serve as a strong constraint on the validity of any model. The particularly vigorous conditions of protein solubilization in SDS that we have used give further confidence that the HP protein band at MW ~ 140,000 does not represent a dimer (or higher aggregate) of peptides with
lower molecular weights. More detailed information regarding the structure of the HP-protein, and the structural relationships involved in its association with the material of the outer bacterial membrane, requires first of all that images of unfixed, unstained protein be obtained at lower electron exposures than those obtained in the present work. The resulting low-dose images should be superimposed and averaged to obtain high resolution data (Kuo and Glaeser, 1975; Unwin and Henderson, 1975). Work of this type can be carried out by developing appropriate cross correlation methods, similar to those described by Saxton and Frank (1977), to superimpose a large number of patches of hexagonally packed material, since the individual patches have too few repeating units to provide the needed high resolution data. Alternatively the procedure for isolating the HP-protein layer may be susceptible to modifications, which would yield larger, single patches with good crystalline order. Finally, it will be necessary to collect image data with tilted specimens and to retrieve the full, three-dimensional structure from a large number of different, tilted views. We wish to thank Prof. R. G. Murray for supplying us with the initial culture of Spirillum serpens strain VHA. We also t h a n k Dr. Ivy Kuo, who conducted preliminary studies in our laboratory; Mrs. Thea Scott-Garner, who assisted with the isolation of the H P layer and with the gel electrophoresis studies; and Dr. Kenneth Taylor for the use of the micrograph from which Figure 2a was extracted. This research was supported by the Department of Energy contract # W-7405-ENG-48 and NIH Grant # G M 23325. REFERENCES
1. AEBI, U., SMITH, P. R., DUBOCHET,J., HENRY, C., AND KELLENBERGER, E. (1973) J. Supramol. Struct. 1, 498-522. 2. AMES, G. F. L., SPUDICH, E. N., AND NIKAIDO, H. (1974) J. Bacteriol. 117, 406-416. 3. BUCKMIRE, F. L. A., AND MURRAY, R. G. E. (1970) Can. J. Microbiol. 16, 1011-1022. 4. BURKMIRE, F. L. A., AND MURRAY, R. G. E. (1973) Can. J. Mierobiol. 19, 59-66. 5. BUCKMIRE,F. L. A., AND MURRAY,R. G. E. (1976) J. Bacteriol. 125, 290-299.
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6. CHESTER, I. R., AND MURRAY, R. G. E (1975) J. Bacteriol. 124, 1168-1176. 7. DERoSIER, D. J. ANDMOORE, P. B., J. Mol. Biol. 52, 355 (1970). 8. FAIl'BANKS, G., STECK, T. L., AND WALLACH, D. F. H. (1971) Biochem. 10, 2606-2617. 9. GRANO, D. A. (1979) Ph.D. Thesis, University of California, Berkeley. 10. KISELEV, N. A. AND KLUG, A. (1969) J. Mol. Biol. 40, 155-171. 11. Kuo, I. A. M., AND GLAESER, R.M. (1975) Ultramicroscopy 1, 53-66. 12. LAEMMLI,U. K. (1970) Nature 227, 680-685. 13. MELLEMA, J. E., AND VAN DEN BERG, H. J. N. (1974) J. Supramol. Struct. 2, 17-31. 14. MURRAY, R. G. E. (1963) Can. J. Microbiol. 9, 381-392.
15. ROSENBUSCH, J. P. (1974) J. Biol. Chem. 249, 8019-8029. 16. SAXTON, W. 0., AND FRANK, J. (1977) Ultramicroscopy 2, 219-227. 17. SLEYTR, U. B. (1976) J. Ultrastruct. Res. 55, 360377. 18. TAYLOR,K. A. (1978) J. Microscopy 112, 115-125. 19. TAYLOR, K. A., AND GLAESER, R. M. (1976) J. Ultrastruct. Res. 55, 448--456. 20. TAYLOR, K. A., GRANO, D. A., AND CHIU, W. (1976) Proceedings 34th Annual Meeting EMSA, 136-137. 21. THORNLEY, M. J., GLAUERT,A. M., AND SLEYTR, U. B. (1974) Phil. Trans. R. Soc. Lond. B. 268, 147-153. 22. UNWIN, P. N. T., AND HENDERSON, R. (1975) J. Mol. Biol. 94, 425-440.
