Paracrystalline aggregates of bacteriochlorophyll protein from green photosynthetic bacteria

ARCHIVES

OF

BIOCHEMISTRY

AND

Paracrystalline

130, 140-147 (1969)

BIOPHYSICS

Aggregates from

RODNEY

Laboratory of Physical National

Green

of Bacteriochlorophyll

Photosynthetic

Protein

Bacteria

A. OLSON, WILLIAM H. JENNINGS, AND CHARLES H. HANNA Biology,

Institutes

National Institute

of Arthritis and Metabolic Diseases, Bethesda, Maryland SO014

of Health,

Received October 16, 1968; accepted November

9, 1968

Paracrystalline aggregates of bacteriochlorophyll-protein are observed in preparations of the mother liquor from which crystals have been obtained. These aggregates are confocal in texture but are rigid and fragile as opposed to the classic examples of confocal smectic “liquid crystals.” Observations of birefringence and dichroism indicate that bacteriochlorophyll is molecularly oriented in a filamentous fine texture that constitutes each ordered domain of the aggregates. Electron microscopy of these aggregates shows that this texture is composed of tubules. These tubules are more or less parallel to each other within the small confines of each domain but their lateral spacing is variable and random. Cross sections of the tubules show a hexagonal array of electron-dense elements surrounding an electron-transparent channel. This array seems congruous but not identical to hexagonal units in the lattice observed in cross sections of bacteriochlorophyll-protein crystals. The macromolecular arrangement is considered in terms of the lateral bonding sites in bacteriochlorophyll-protein paracrystals.

The water-soluble complex of bacteriochlorophyll-protein (Bchl-P) isolated from the green photosynthetic bacterium Cs. e&y&urn by Olson at the Brookhaven National Laboratory, has been studied extensively (l-7). The complex crystallizes in a hexagonal habit with pinacoid planes forming six-sided pyramidal ends (6). These crystals, which can attain dimensions of over 75 tirn in thickness and up to 700 pm in length, have been studied by X-ray diffraction and electron microscopy (6). X-ray measurements of the crystals reveal the hexagonal space group, P63, with an “elemental” unit cell containing 6 macromolecules (120 bacteriochlorophyll moles). Electron microscopy of glutaraldehyde-fixed crystals shows what appears to be the crystal lattice. Sections cut parallel to the long axis show electron-dense striations parallel to the axis. Sections cut perpendicular to this axis show a hexagonal array of electron-transparent holes. Olson et al. (6) interpreted

these holes and striations as channels parallel to the c axis at each corner and one through the center of each unit cell.’ They proposed a macromolecular shape approximating a prolate ellipsoid (8.8 X 6.5 nm) consistent with macromolecular volume and considerations of crystal packing. Investigation of the optical properties of single Bchl-I’ crystals (7) supports and extends the above findings. The absorption spectrum of the crystals is essentially that of the complex in solution. The spectrum is weakly polarized at both the 603-nm (D = l/1.2) and the 809-nm (D = 1.3) absorbance maxima. This dichroism, negative at 603 nm and positive at 809 nm with respect to the optic axis, indicates that the Bchl-binding sites are oriented. In the course of our studies of Bchl-P 1The optic axis and the crystallographic c axis are parallel to the longest geometrical axis of the Bchl-P crystal. 140

BACTERIOCHLOROPHYLL

crystals (7) several solutions of Bchl-P complex produced paracrystalline aggregates. These aggregates consisted of Bchl-P and were optically anisotropic. This new form differs in shape and texture from that of the crystals. The aggregate texture could be recognized as long structural elements not quite resolvable by light microscopy. The aim of this study is to compare the structure and macromolecular arrangement of these two forms in order to develop information about the bonding sites in Bchl-P. MATERIALS

AND

METHODS

Cryslals. Purified Bchl-P was prepared from Cs. ethylicunt as described previously (I). Crystals were grown in mother liquor with 0.01 >f phosphate buffer (ph 7.8), 1.0 M N&l, and 0.6 M (XHJ$Oh as described by Olson. The irregular paracrystalline aggregates appeared spontaneously in mother liquor preparations of similar composition. Optical anisotropy. rl Reichert hIeF metallographic microscope (polarizer and analyzer are Nicol prisms) was used for observations of birefringence and dichroism. The anomalous dispersion of birefringence was measured with a Berek compensator (E. Leitz). Dichroism was measured by a method described in Ref. 7 and a Varo image converter extended the observations into the infrared. Electron microscopy. The large crystals used in this study were fixed with glutaraldehyde as in Ref. 6. The much smaller paracrystalline aggregates were fixed in 37; glutaraldehyde in phosphate buffer (pH 7.8). Both types of fixed material were dehydrated through a graded series of ethanolwater mixtures and embedded in epoxy resin. Sections cut with a diamond knife were 50-80 nm in thickness. Prior to examination in the electron microscope, the sections were stained successively with solutions of uranyl acetate and lead citrate. Crystals of the dyestuff Indant,hrene Olive TWP prepared as described in Ref. 8 have a lattice spacing of 2.49 Ilrn which provides a convenient and accllrate calibration scale for the electron micrographs. RESULTS

ilND

DISCUSSION

In our laboratory the paracrystalline forms of Bchl-P appeared spontaneously in preparations of mother liquor after filtration to remove large crystals and the addition of (SH&S04. After several days small blue clumps appeared in the shape of randomly interlocked cones. As these confocal groups

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increased in size and optical density a few crystals appeared. Over a period of several weeks more crystals appeared until they approximately equalled the number of paracrystalline aggregates. This population of crystals apparently was not produced at the expense of the aggregates. The interesting feature of the paracrystalline aggregates is their resemblance to polymer spherulites and to the confocal texture of smectic type “liquid crystals” formed by certain other unrelated materials (see Ref. 9). There are differences, however, in their reaction to crushing or shearing forces. Confocal textures of liquid crystals are nonrigid and form between confining surfaces as space-filling elements. If parallel confining surfaces (i.e., slide and cover glass) are moved with respect to one another, the smectic texture is altered but no fracturing occurs. Even though crushed or sheared, the liquid crystal readily forms a new confocal texture. On the other hand, B&-P focal conic structures are not attached to the confining surfaces of the preparation as spacefilling elements and remain intact between shearing surfaces. The paracrystals arc, therefore, not liquid crystals in t’he classic sense. An example of a typical paracrystalline aggregate of Bchl-P is shown in Fig. 1A. The figure also shows the characteristic fine texture of each conic element in an aggregate group of paracrystals. This texture is brush-like and radiates from the apex of each confocal domain. The over-all texture can be observed best at the 603-nm absorption peak for Bchl-P but the individual contributing elements are not quite resolvable by light microscopy. Optical properties. The spectral absorbance of Bchl-I’ parncrystalline aggregates approximates that of the crystals. It is characterized by a strong absorption maximum at SO9 nm with a shoulder near 750 nm. At GO3 nm there is a second maximum with a shoulder near 544 nm. Maximum absorbantes for aggregates like those in Fig. 1A fall in the range of 0.3-1.2 for 603 nm and 0.8-1.7 for SO9 nm. The absorbance is markedly polarized at 809 nm and can be easily observed with the infrared image converter. This is demonstrated by infrared

142

OLSON, JENNINGS,

AND HANNA

FIG. 1. A. Light micrograph (600 nm) of Bchl-P paracrystalline aggregates. B and C. Birefringence of (A) where the electric vector of the light transmitted by the analyzer and the polarizer is indicated by the arrows. X 415.

FIG. 2. A and B. Dichroism indicate

the electric

vector

of Bchl-P paracrystalline of the light. X 350.

photomicrography in Fig. 2A and B. Polarized light in the spectral region of 809 nm is preferentially absorbed parallel to the axis of the filamentous texture. Measurement of this preferential absorption yields a positive dichroic ratio: D = 1.50 (average of six measurements). In the AOS-nm spectral region the absorbance is considerably less polarized, D = l/1.09. These observations indicate that the 809-nm electronic transition oscillators have a slightly greater orientation parallel to the direction of filamentous texture than the 809-nm oscillators in the crystals have to the direction of the optic axis. Conversely, the degree of orientation of the 603-nm oscillators in the Bchl-P aggregates appears to be slightly less

aggregates

at 809 nm. The arrows

than that in the Bchl-P crystals. The birefringence of Bchl-P aggregates is weak and of the same order and sign as that of Bchl-P crystals. Figure 1B and C demonstrates the optical anisotropy of the aggregates in white light. The arrows represent the electric vectors of the light transmitted by each of the Nicol prisms. When the electric vector is parallel to the direction of the filamentous texture of a domain, maximum extinction occurs (lower left, large domain in Fig. 1C). Conversely when the electric vector is 45” to the direction of the filamentous texture, maximum brightness occurs (same domain in Fig. 1B). The filamentous texture of the aggregates thus exhibits parallel extinction. In white light the retardation color of the

BSCTEI~IOCHLOROPHYLL

FIG. 3. Electron micrograph tous elements. X 6500.

of a paracrystalline

Bchl-I’ aggregates is bluish-white in contrast to the orange color observed in the crystals. This difference is reflected in the character of the anomalous dispersion of birefringence. In Bchl-I’ aggregates the anomalous dispersion has a constant positive value in the visible region with a marked normal pattern in the SOS-nm region. This indicates that the SOS-nm trunsition oscillators are preferentially oriented in the direction of the filamentous texture. It also confirms the small preferential orientation of the 003-nm oscillators. Electrm ~rnicroscopy. Even at lo\\- power (X 1700) the electron microscope resolves the filamentous texture of the Bchl-I’ paracrystalline aggregates (Fig. 3). The aggregate is composed of loosely packed filaments arranged in several different domains of orientation. Each of these domains cow sists of the conical filamentous textural

143

PROTEIK

aggregat,e showing

domains

of filamen-

domains seen in whole preparations observed by light microscopy. The figure also shows the demarkation boundaries between different domains of filament orientation. The upper right portion of the specimen section was cut perpendicular to the direction of filament orientation and shows a cross-sectional aspect of the filaments. The spacing of these filaments is random with no indication of systematic lateral articulation. Voids and interstices are spacious compared to the areas occupied by the filaments. In the central vertical portion of the specimen a domain has been cut parsllel to the direction of filament orientation. Here the individual filaments are undulatory and irregular. The specimen section also shows zones of intercalation of textural domains. Higher magnification reveals structurnl details of the filaments. A transverse section of the filaments (Fig. 44) show that each contains an

144

OLSON, JENNINGS,

AND HANNA

FIG. 4. A. Electron micrograph of filamentous elements of Fig. 3 in cross section showing a hexagonal array of cusps about an electron-transparent hole. B. Elements of Fig. 4A viewed in a longitudinal section showing long tubular structures. X 13700.

electron-transparent hole. A section containing a longitudinal array of the filaments (Fig. 4B) indicates that the filaments are electron-dense tubules. Closer examination of the transverse sections of these tubules shows that they are composed of electrondense elements articulated in a hexagonal array. Neither the shape of these elements nor their mode of articulation can be resolved. Part of the difficulty lies in the irregularity and nonparallelism of the oriented tubules in a given section. However, the tubules in cross section appear to have projections or cusps radiating outward from each apex of their hexagonal array. While in many instances some of these cusps appear to be touching those of close neighbors, many do not. Since the majority of the cusps are free, it is assumed that they are not optimal attachment sites. It is interesting to compare the structure of these tubules of Bchl-P aggregates with that of the elements which appear to constitute the lattice array in Bchl-P hexagonal crystals. Because the crystal electron micrographs of Ref. 6

were prepared by slightly different techniques than those used here, we have included in Fig. 5 (5B and C) electron micrographs of Bchl-P crystals cut in our laboratory. Figure 5A is an enlargement of a portion of Fig. 4A showing the hexagDna1 tubules in cross section at the proper scale for comparison. The insets in the lower right hand corner of this figure and of 5B are not parts of the electron micrographs and will be discussed later. Figure 5B shows a section of a crystal which was cut perpendicular to the long axis of the crystal. Prominent is the regular array of equidistant holes extending in vertical rows. While the elements surrounding the holes or channels cannot be resolved in a regular pattern, they form a hexagonal array as shown encircled in the figure. This array is repeated throughout the crystal cross section and is the smallest structural element that can be resolved in such an electron micrograph. The average repeat distance between parallel rows of channels ranges from 9.0 to 11.1 nm and the average channel-to-channel distance is 9.2,5

FIG. 5. A. Elements in Fig. 4A at proper scale for comparison with crystal micrographs. [Refer to text for description of inset here and in 5B]. B. Electron micrograph of a Bchl-P crystal sectioned perpendicular to the long (c) axis showing circumscribed hexagonal array of six electron-transparent holes. C. Electron micrograph of a Bchl-P crystal sectioned parallel to the long (c) axis showing the electron-transparent striations along this axis. X 246000.

in macromolecular arrangement. This differnm. The diameter of each channel is approximately 7.0 nm (see Ref. 6). A section cut ence may be resolved by a study of macromolecular models of the crystal. parallel to the long axis of a Bchl-P crystal The arrangement of macromolecules which is shown in Fig. 5C. Some similarity between the elements of the crystal lattice and the forms the crystal lattice has been considered hexagonal tubules can be observed in Fig. in detail by Olson et al. (6). From their dia5A. The small portion of the crystal cross grams we have assembled a three-dimensection encircled in Fig. 5B contains a hexag- sional model of the crystal lattice. Although onal element of structure which is some- the macromolecules are postulated as ellipwhat similar to the transverse sections of soidal (with major axis parallel to the c tubules sh0hT-n in Fig. 5A. There are, how- axis) the styrofoam spheres used for the ever, some obvious differences. The en- model gives a good representation of the circled portion of Fig. 5B contains seven macromolecules viewed along the c axis. channels equally spaced, six of which are Figure 6A is a photograph of this model arrayed hexagonally around a central one. taken nearly parallel to the c axis. This view No such hexagonal array of channels ap- of the model shows the hexagonal array of pears in the cross sections of the tubules in six channels about a central channel (cirFig. 5A. Here the cusps which extend about cumscribed in Fig. 6A) which compares with a large central channel form the hexagonal the electron-transparent holes in Fig. 5B. array. Comparison of the dimensions of the Around each channel the macromolecules crystal elements and those of the tubules are hexagonally displayed. This arrangeindicates another difference in constitution. ment provides eight points of contact or In the tubules the central channel is much attachment sites for each macromolecule larger than those in the crystal. The average to bond to its nearest neighbor; two of these diameter of 24 channels measured in Fig. 4A bonds would be longitudinal between the is 12.9 nm. This is nearly twice the diameter apices of the ellipsoids (parallel to the c of the channels in the crystal. lcurthermore axis) and the other six more or less lateral the over-all dimension of the tubules meas- (perpendicular to the c axis). The filaured from cusp to cusp is 41.8 nm. This di- mentous aggregates are characterized by mension is greater than that which could be loose lateral bonds compared to the firm ascribed to the hexagonal structure containlateral bonds of the crystal. This difference ing seven holes (see encircled area of the may arise from a difference in the composicrystal, Fig. 5B). The Bchl-P paracrystals, tion or the arrangement of the macromolethen, must differ from the Bchl-P crystals cules, or perhaps both. The common origin

146

OLSON,

JENNINGS,

AND

HSNNA

FIG. 6. A. Model of the crystal lattice showing circumscribed hexagonal array of six holes and an isolated grouping of three longitudinal rows of spheres which surround one half of each channel. B. Model of a oaracrvstalline aggregate filament showing the large channel surrounded by six cusp-like projections.

of both filaments and crystals from normal onal filament aggregates (channel diameter = 12.9 nm; dimension across mother liquor and the similarity of their spectral properties indicate that a major cusps = 41.8 nm). The cmorrespondence of alteration of the Bchl-P macromolecule is un- these models with their counterparts illuslikely. In constructing a filament model, the trated by the electron micrographs is shown by the insets in the lower right hand corner numerous possibilities have been restricted of Fig. 5A and B.2 The inset in Fig. 5A shows to those derived from a simple rearrangethat the macromolecular configuration of ment of the elements of the crystal lattice. This approach minimizes the differences in the model in Fig. 6B is an acceptable intsrpretation for that of the filament cross articulation of the macromolecules between section observed by electron microscopy. the two forms. If we examine the arrangeReplication of the macromolecular strucment of macromolecules which make up the ture illustrated in Fig. 6B appears to be wall of each channel, we find a repeating unit consisting of three longitudinal rows of favorable in the longitudinal direction but unfavorable in the lateral direction. Longispheres around one half of each channel. Such a unit is shown removed from the model tudinally, replication is nearly the same as at the top of Fig. 6A. In Fig. 6B, six of these that found in the crystal lattice. Laterally, however, extension of the adjacent cusps units have been assembled into a hexagonal array with one large channel and six cusp- by added macromolecules to form channels would involve symmetrical elements conlike projections. The formation of this array requires that two lateral bonding sites taining ten-membered rings. This does not (associated with one macromolecule in the appear to be compatible with the growth of a group of three) be rotated about the c axis. 2 The Styrofoam sphere models when photoThe scale diameter of the channel in the graphed in transmitted light produce density patmodel is 15.5 nm and the dimension across terns comparable to the electron micrographs. The the opposite cusps is close to 43.7 nm. These photographic negatives were projection printed dimensions are commensurate with those with a diffusion screen at the proper scale to simtaken from electron micrographs of hexag- ulate the elect,ron micrographs.

BACTERIOCHLOROPHYLL

system which is basically hexagonal. The minor rearrangement of the lateral bonds in the Bchl-P filaments may result in new constraints on the Bchl molecules. These may be reflected by the differences in optical anisotropy between the crystals and the paracrystalline aggregates. The dichroism of the aggregates, slightly greater at 809 nm and slightly smaller at 603 nm, indicates a change in the average position of the Bchl molecule from that in the crystal. The magnitude of this change is so small (as calculated by pro cedures detailed in App. II, Ref. 7) that the assumption of no major alteration of the Bchl-P macromolecule between the crystal and the paracrystalline aggregate appears to be valid. The confocal structure of liquid crystals results from a molecular arrangement in the lowest energy state. Similar arguments might be made for the confocal structure of Bchl-P aggregates; how-ever, the friability of the aggregates indicates that in addition to strong longitudinal bonds a limited lateral bonding also exists. As seen from the electron micrographs, the lateral bonds of the aggregates are not sufficiently strong or perhaps suitably located to permit lateral extension. Some cases of lateral articulation at the ends of cusps (see Fig. 4A) give indication of the limited lateral bonding. The complex of bacteriochlorophyll and protein is obtained from living material and some of its properties may be similar to the properties of the complex in viva. Particularly interesting is the potential of the complex to form several types of replicating structures. It is also significant that the pigment Bchl, a photosynthetic pigment, is oriented within the organized protein struc-

l-17

PROTEIN

ture. Our earlier work with chlorophyll a in the lamellae of living cells indicated that a major component of this pigment was highly oriented with respect to the lamellae (10). We said then that the oriented pigment should be related to the orderly fine structure of the lamellae and that the orientational aspects of the protein moiety in chloroplasts should command attention rather than those of chlorophyll. The present study probes the orientational aspects of a macromolecular complex of protein and pigment from a photosynthetic system and provides insight concerning the articulation of these into extensive structures. REFEREXCES 1. OLSON, J. Biophys. 2. OLSON, J. Vernon Academic 3. OLSON, J.

4. 5. 6. 7.

8. 9.

10.

RI., AND ROMANO, C. A., Biochim. Acta 69, 726 M., in “The and G. R. Press, New

(1962). Chlorophylls” (L. P. Seely, eds.), p. 413. York (1966). XI., FILMER, D., RADLOFF, R., ROMANO, C. A., AND SYUESMA, C., in “Bacterial Photosynthesis” (H. Gest, A. San Pietro, and L. P. Vernon, eds.), p. 423. Antioch Press, Yellow Springs, Ohio (1963). GHOSH, A. K., AND OLSON, J. hl., Biochim. Biophys. Acta 162, 135 (1968). THORNBER, J. P., AND OLSON, J. &I., Biochemistry 7, 2242 (1968). OLSON, J. M.,KOENIG, D.F., AND LEDBETTER, M. C., Arch. Biochem. Biophys. in press. OLSOX, R. A., JENNINGS, W. H., AND OLSON, J. M., Arch. Biochem. Biophys. in press. LABAW, L. W., J. Applied Phys. 36, 3076 (1964). GRAY, G. W., in “Molecular Structure and the Properties of Liquid Crystals,” p. 18. Academic Press, New York (1962). OLSON, R. A., JENNINGS,W. H., AND BUTLER, W. L., Biochim. Biophys. Acta 66, 318 (1964).