Polymorphic assemblies of double strands of sickle cell hemoglobin

,J. Mol. Rio/. (1981) 153. 1011-1026

Polymorphic Assemblies of Double of Sickle Cell Hemoglobin Manifold

Pathways

THOMAS E. WELLEMS, lkpartment 7%

(Hw&~d

l~ttiwrsity

of Deoxyhemoglobin ROBERT

Strands

S Crystallization

,J. VASSAR AND ROBERT ,JOS~CPHS

of Biophysics attd Theoretical Hioloyy of Chicago, Chicago, 111. 60637, I’.S..-l

31 Octolwr 1980. attd itt rrvisrd form

2 Frbruary

1.981)

(‘rystallization of sickle cell hemoglobin proceeds by distinctive pathways which depend upon thr pH and the ionic composition of the crystallizing milieu. The pathways differ in that after fibers form they associate into different intermediatis which then cryst,allize. We term the pathways “high pH” and “low pH”. The value of the transition pH Mween the high pH and low pH pathways depends upon the specific ionic species present in the hemoglobin solution. Over the pH range studied the mechanism of crystallization is pa-dependent but the structure of the crystals ultimately formed is not. In this paper we describe t,wo newly discovered intermediates involved in the crystallization of droxyhemoglobin S via the low pH pathway. The first of these consists of a class of particles we call macrofibers. Optical diffraction patterns of fibers and macrofibers have similar intensity distributions and layer-line spacings suggesting that macrofibers and fibers are assembled from a common structural unit which \c’e take to be the Wishner-Love double strand. The second new structure is a paracrystalline form of deoxyhemoglobin R. The paracrystal is built from layers of double strands of molecules in an arrangement similar to that within the crystals. Optical diffraction of electron micrographs of paracrystals reveals that longitudinal disorder is present between double strands. I’rojections of the electron density down the c axis of the crystal provide images very similar to those in electron micrographs of negatively stained paracrystals. The patterns appearing in the paracrystal due to the disorder can be fully simulated by shifts between the layers of double strands.

1. Introduction Sickle cell hemoglobin differs from normal hemoglohin because of a mutation which replaces glutamate hy saline at the sixth position of each /I chain. This single hydrophobic amino acid substitution greatly reduces the solubility of the droxygenated form of the molecule (I’erutz et al., 1951). Reduction of the solubility of hemoglobin results in the formation of long rigid fibers under physiological conditions. Bundles of these fibers are responsible for the sickling of red Mood cells and the clinical manifestations of sickle cell anemia. 1011

1012

T. E. M’ELLEMS.

R. .I. VASSAR

AND

R. dOSEPHS

Wishner et al. (1975a) have found that deoxyhemoglobin S crystals suitable for X-ray analysis grow in citrate/phosphate buffer and polyethylene glycol at an acid pH (5 to 6). The structure of these crystals has been determined to 3 A (Wishner et al., 1975h). The deoxyhemoglobin S molecules within the crystal are arranged in double strands which are stacked in layers alternately running in opposite directions. In each double strand the molecules are interlocked and staggered by 32 A so an approximate S-fold screw axis runs along the double strand. Slowly stirring solutions of deoxyhemoglobin S near physiological conditions of pH and ionic strength will also induce crystallization (Pumphrey & Kteinhardt, 1976,1977). Our investigations of this phenomenon have indicated that crystallization occurs by a process in which fibers align into fascicles and then fuse (Wellems & Josephs. 1979). We have further found the space group and unit cell dimensions of these crystals to be indistinguishable from those of crystals grown in polyethylene glycol and citrate/phosphate buffer at pH 5 to 6 (acidic I’EC:). The observation that fibers fuse to form crystals implies that the molecular arrangement of the fibers must be closely related to that of the crystal. Within the crystal the predominant packing forces reside in the molecular double strands. suggesting that the structure of the fiber can be analyzed in terms of modified packing of the double strands. For crystals to form from fascicles, the fibers comprising the fascicles must be able to fuse regardless of the direction they run in the fascicle. This consideration has led us to propose that the fibers are not polar (i.e. they have no directionality). ?\‘or is there any experimental evidence supporting the view that the fibers are in fact polar. Since th(x double strands are polar this implies that equal numbers of double strands must run in opposite directions along the length of the fiber. That’ is, the fiber is built from pairs of antiparallel double strands. Eight double strands produce fibers w&h diameters close to those experimentally observed, and we have thereforcl proposed that the fiber consists of four double strands running in each direction along its length (Wrllems & Josephs, 1979: Wellems, 1980). This view of the fiber structure differs from that proposed by Dykes et al. (1978) on the basis of their electron microscope studies. The major point of divergence is that the model of the fiber put forlvard by Dykes et al. (1978) contains seven double strands. If the deoxyhemoglobin 8 fiber comprised seven double strands, then it would necessarily be polar. Crystallization could not’ then proceed by the process of alignment and fusion we have documcnt)ed. X more complex mechanism \vould need to be invoked. For instance, one might postulate that crystallization occurs in t\vo steps: the first step would involve pairing of fibers of opposite polarity. thus producing non-polar pairs, follo\ved by crystallization. Another hypothetical mode of crystallization might involve either loss or gain of double strands to form nonpolar fibers prior to crystal formation. Support for crystallization involving polar fibers could come from observations of either fiber pairing or double strand exchange during crystallizat,ion. In the present work we describe further the phenomenon of crystallization of deoxyhemoglobin S in stirred solutions. The initial event is fiber formation. Crystallization then occurs by pathways which depend upon pH. The pathways differ in that after fibers form they associate further to form distinctive

ASSEMBLIES

OF DEOSY-HbS

DOUBLE

STKANI)S

1013

intermediates prior to crystallization. We refer to the pathways as “high pH” and “low pH”. A transition pH exists between the high pH and low pH pathway. the value of \vhich depends upon the particular ionic species present. For example, in 50 miv-phosphate the transition pH is 65 kO.2 ; in 50 mM-Tris it is pH 7.1 f@2. The lo\v pH pathway involves fibers associating to form particles which have not hitherto been described. We call this new kind of particle a macrofiber. Optical transforms of fibers and macrofibers have similar intensity distributions and layerlint, spacings suggesting that the two types of particles have similar structures. I!lacrofihrrs in turn develop into paracrystals. The paracrystals subsequentI> anneal to form \vell-ordered crystals having the same space group and unit crll tlimrnsions (as ol)served at electron microscope resolution) as crystals grown in acidic I’!& : ()ptical diffraction of electron micrographs of paracrystals shows that they are made up of layers of double strands in an arrangement similar to that I\-ithin t)he crystal. hut that poor registration exists between the layers of double et al. strands. The crystal structure data of Wishner et al. (1971ia) and hluirhead (1967) have been used to compute the projection of the electron density down thr c axis of the crystal. The density profile obtained is very similar to that observed irh c~lectron micrographs of negatively stained paracrystals. The appearance of micrographs of the paracrystals is accounted for in terms of a translational shift of the double strands along the a axis of the crystal.

2. Methods of Experimental Procedure (a) Solution

compositions

and

stirring

rxperimuds

Sickl(b cell hemoglobin was purified, stored, and prepared for microscopy previously (Wellems & Josephs. 1979; Wellems. 1980).

as described

(I)) Electron microscopy ad optical diffraction The preparation of negatively stained specimens and electron microscopy were carried out as described previously (Wellems & Josephs, 1979). Optical transforms were taken using a folded optical diffractometer. Measurements from the micrographs and from the transforms were made using a Nikon model 6C microcomparator. (c) Computer-generated

projections

of electron density

The projections presented in this work were calculated using the atomic co-ordinates of deox~hemoglobin reported 1)~ Muirhead et al. (1967) and the molecular translations and rotations given by Wishner et al. (197.‘). Each unit cell contains 4 molecules. Molecules 1 and 2 are members of one double strand passing through the cell and running along the a axis. Molecules 3 and 4 are members of a second double strand running in the opposite direction. The co-ordinates of each projected atom were calculated and a Gaussian distribution weighted by the electron density of the atom was centered on these coordinates. The contribution of this distribution to the nearby grid points of an array representing the projected electron density was then computed. The final projection of the unit cell was built up by repeating this process for all atoms. A “smoothed” projection was obtained by Fourier transformation of the density distribution, multiplication by a Gaussian smoothing function in reciprocal space, and reverse transformation. The Gaussian function was of the form P-(~*~~)‘,where R is the reciprocal space dimension in A-‘. This smoothing function produced projections having a resolution comparable to that of electron

T. E. WELLEMS,

R. J. VASSAR

AND

R. JOSEPHS

b)

FIG. 1. Electron micrographs of negatively stained macrofibers of deoxyhemoglobin 8. (a) A macrofiber having 6 rows grown in 50 mM-phosphate. (b) A macrofiber having 4 rows grown in 50 mMphosphate. (c) A macrofiber having 5 rows grown in 50 mnr-Tris. (d) A macrofiber having 6 rows grown in 50 mnr-Tris.

hSSEMBLIES

OF DEOSY-HbS

DOIJBLE

STRANI)S

(d)

1016

‘I’. E. WELLEMS,

R. .J. VASSAR

AND

R. JOSEPHS

micrographs and avoided a step function cut-off of the frequency spectrum which could generate artifacts in the projected image. Arrays having double strands shifted with respect to one another were generated by incrementally shifting molecules 3 and 4 along the crystalline a axis. Each projected density was recorded with an FR-80 film plotter (Information International, Inc.) using spot intensity control. The density was repeatedly plotted in each direction so that the projected unit cell was displayed in a 2-dimensional lattice. All images were recorded on Kodak Dacomatic G film.

3. Intermediates in the Crystallization

Process are pH Dependent

The first step in the crystallization of deoxyhemoglobin S is fiber formation. The fibers then assemble to form larger aggregates whose structure depends upon the pH. We have characterized a high pH pathway and a low pH pathway. The value of the transition pH between the two pathways depends upon the ionic species present. In 50 m&r-phosphate the transition pH is 6.5 kO.2 ; in 50 mm-Tris it is pH 7.1 kO.2. When crystallization proceeds by the high pH pathway, fibers form fascicles which are present for 12 to 48 hours before conversion to crystals takes place (Wellems & Josephs, 1979,198O). The low pH pathway involves formation of particles which we term macrofibers (Fig. 1). Macrofibers are a relatively stable class of particles and fill the solution volume for several hours before further developments in the transition occur. The macrofibers convert into paracrystalline slabs which typically range from 1 to lOO~m* in area. Variations in temperature (25 to 35°C) or stir rate (2 to 12 cycle/s) alter the kinetics but have not been observed to affect the intermediate structures that form during the crystallization process. If stirring is stopped Earacrystal formation is delayed for one to three weeks. In order to test the stability of macrofibers relative to fascicles, we have carried out parallel experiments in which two solutions, one containing 170 mg hemoglobin S/ml in 50 mM-Tris, 61 m&r-EDTA (pH 6%), and the other containing 170 mg hemoglobin S/ml in 50 mM-phosphate, 91 mM-EDTA (pH 6.8). were simultaneously stirred and deoxygenated on the same magnetic stirrer. As expected, macrofibers developed in the first solution while fascicles were found in the second solution. When portions of the solution of macrofibers (up to 10% by vol.) were added to the solution of fascicles the macrofibers rapidly dissolved, indicating that the macrofibers are not stable relative to fibers in phosphate buffer at pH 6%.

4. Macrofibers of Deoxyhemoglobin

S

The macrofiber is an intermediate having structural features in common with both the fiber and the crystal. Indeed its structure is of interest just because it is an intermediate which links the fiber with the crystal. Figure 2(a) presents a micrograph of a macrofiber formed in 50 m&r-Tris. This particle has a diameter of 510 A across the narrow region and 660 A across the wide region. The variation in diameter is periodic and is about 5100 A, but this value often varies by as much as

ASSEMBLIES

OF DEOSY-HbS

DOlTBLE

1017

STKANIIS

(b)

(4

(d)

(20 Ar’ FIG. 2. Enlarged micrograph showing a stat-kch of a mat arofiber and its optical transform. (a) The arrow regions of the specimen reveal 5 rows in projection. A fret having a period of 64 A runs along the NW. (b) Optical transform of a length of tt Cs specimen spanning one repeating segment; (c) the ransform of the narrow region ; (d) the trwsfc mn from the wide region.

1018

T. E. WELLEMS,

R. J. VASSAR

If: 300 a from macrofiber to macrofiber

AND

and occasionally

K. JOSEPHS

even along the length

of a

single particle. Different projections of the structure of the macrofiber are apparent along its length. Wide regions of the macrofiber display prominent 32 a striations which run normal to the long axis. In some areas the striations show two discontinuities as they cross the macrofiber (see the region indicated by the arrow in Fig. 2(a)) thus leaving the impression that along this projection the structure is divided into three sections of approximately equal width. A second set of striations which run nearly parallel to the long axis is also present. These striations have a side-to-side period of 54 8. The narrow region of the macrofiber is divided into five rows 95 A + 8 A wide. The central row displays a pattern which can be approximately described as a fret or zig-zag repetition of short segments intersecting at nearly right angles which repeats every 64 A. Figure 2(b) shows an optical transform of one repeating segment of the micrograph in Figure 2(a). A prominent meridional reflection is present at 32 A, a series of less intense reflections runs across the 64 A layer-line, and near-equatorial reflections occur at axial spacings of about 400 to 700 A. We have been able to correlate some of the features of the macrofiber structure with features in the optical transforms of wide and narrow stretches of the macrofiber. The intensities of the near equatorial reflections are greatly diminished in optical transforms of the narrow section (Fig. 2(c)) relative to transforms of the full repeat. In contrast the 64 A layer-line is relatively more intense in transforms of the narrow section and derives from fret patterns generated by five rows running at an angle to the axis. Figure 2(d) sho\vs the transform of the wide stretch of the macrofiber. Here the near equatorial reflections are more intense than in the transform of the full repeat; they derive from the nearly axial 54 A striations seen in the micrograph. Since the reflections are slightly off the equator the striations must be nearly, but not quite, parallel to the particle axis. The interior reflections on the 64 w layer-line are very weak in transforms of the wide section further indicating they originate from structures most readily evident in the narrow aspect of the macrofiber. All regions of the macrofiber give rise to a strong 32 L%meridional reflection which indicates a subunit rise of 32 A along the length of the particle. Optical diffraction patterns of fibers and macrofibers have similar intensity distributions and layer-line spacings (Fig. 3). Patterns from both structures are characterized by near equatorial reflections at a radial spacing of 54 8. Higher order layer-lines index on a 64 a repeat. Transforms of both particles display a prominent meridional reflection at an axial spacing of 32 A. These similarities attest to the close structural relationship of the two kinds of particles.

5. Paracrystals and Crystals of Deoxyhemoglobin

S

Once crystal development has begun the macrofibers are quickly replaced by paracrystals of roughly 1 to 100 pm2 in area such as those shown in Figure 4. A fret similar to that present in the narrow region of the macrofiber in Figure 2 is evident in the region of Figure 4(a), indicated by the arrow. The fret is well developed in

ASSEMBLIES

OF DEOXY-HbS

-

328

DOUBLE

STRANDS

-

Macrofiber FIG. S. A comparison of the optical transforms spacings and intensity distribution.

IO19

I--

Fiber of fibers and macrofibers.

Note the similarities

in the

each row, but disorder in the registration of the rows is evident in the micrograph. Disorder is also indicated by the optical transform of the micrograph (Fig. 5(a)). The reflections in the transform fall sharply on the 64 a and 32 .A layer-lines. but the intensity distribution along the layer-line shows no indication of lattice sampling except on the equator where the reflections show a periodicity of about 90 A. corresponding to the distance between rows in the imaget. Figure 4(b) presents a micrograph of a specimen in which crystalline order is extensively developed. The herringbone pattern in the image consists of segments arranged in interlocking stacks. The segments in adjacent stacks slant in opposite directions at about 80” to the longitudinal axis of the stacks. Figure 5(b) shows an optical transform of this micrograph. The reflections on the layer-lines are well defined and fall on a reciprocal space grid identical to that described for crystals of deoxyhemoglobin S (Wellems & Josephs, 1979). Thus to the resolution inherent in the micrographs, the space group and unit cell dimensions are the same as those of crystals grown in acidic PEG. The fret and the herringbone pattern are both present in Figure 4(c); each pattern extending across at most only two or three stacks. Figure 5(c) and (d) shows two exposures of the optical transform of Figure 4(c) ; the former revealing t,he detail in the low resolution layer-lines and the latter the detail at high& resolution. The spacing of the fifth layer-line is 12.7 A. Again the longitudinal registration of the rows is imperfect. Since the fully developed crystalline state has the same structure as the ctystals described by Wishner et al. (1975a,h) we have attempted to account for the t The equator of the transform is the transform perpendicular to the rows. Such a projection does registration of the rows but only their projected density. the projection will have a periodic variation in density will yield the spacing bet.ween rows regardless of their

of the projection of the density onto a line not contain information on the longitudinal If the spacing between the rows is uniform then and the spacing of the reflections on the equator longitudinal registration.

1020

T. E. WELLEMS.

R. J. VASSAR

ANI)

Ii. JOSEPHS

FIG. 4. Micrographs of negatively stained paracrystals and crystals of deoxyhemoglobin S. Micrograph (a) shows a well-developed fret similar to that found in macrofibers (Fig. 2). (b) A specimen having extensively developed crystalline order. A herringbone pattern is present. Micrograph (c) shows an image in which both the herrmgbone pattern and the fret are present.

appearance of the disordered paracrystals on the basis of that structure. In order to do so we have computed the projection of the electron density of the crystal down the c axis. We have also computed projections in which the double strands have been shifted along the crystalline a axis. Figure 6 presents a series of these computed projections. The close resemblance between the herringbone pattern of Figure 6(a) with that of electron micrographs of the negatively stained crystals (Fig. 5(b) and Fig. 9 of Wellems & Josephs, 1979) is immediately apparent, The same herringbone pattern is also evident in the “crossover” region of helical crystals of deoxyhemoglobin S (Fig. 7(c) of Wellems & Josephs, 1979). The pattern is composed of interlocking segments alternating at angles of about k 10” to the b axis. Heavy spots of projected density are present at the ends of each segment. In Figure 6(b) to ( j) the

ASSEhlHI,IES

OF DEOSY-HbS

INI’BLE

.

e-w*

*A.~*

(20 .

1021

STKAXl)S

.c*

*.

‘am .

al-1

-

VII:. 5. Optical transforms of the micrographs shown in Fig. 4. The reflections in the transforms fall sharplv on la.ver-lines at harmonics of 64 A. The transform of the image having crystalline order is shown”in (b). (The reflections fall on a reciprocal space grid identical to that of crystals grown in acidic PM:.) The transform shown in (d) is of the same region as that in (c) but has been exposed to record the higher-order layer-lines. Five layer-lines am evident, corresponding to a resolution of 11.7 A.

registrat,ion between the double strands running through each unit cell is shifted along the n axis by @l unit cell (635 A) in each succeeding frame. As the shift is increased continuity between the segments appears to distort (Fig. 6(b)) and then rupture (Fig. 6(d)). With further shifting of the double strands the segments reappear, displaying a bend in the middle (Fig. 6(i)). A shift of one full unit cell brings the structure into equivalence with the starting structure.

(b)

(cl

(d)

FIG. 6. The crystal electron densitv projected down the c axis (see Methods of Experimental Procedure for details). In order to facilitate micrographs. protein is reprvsent,ed’in white. The shifts are along the a axis and correspond to @l unit cell/frame.

(a)

comparison with the

(e)

ASSEMBLIES

OF DEOSY-HbS

DOC’BLE

S’I’RANI)S

1f )23

The projections of individual double strands can be most easily seen where the shift is sufficient to break continuity of the segments. The projection of each double strand is a fret with periodic spots of high density. Adjacent double strands run in opposite directions, as can be appreciated from the positions of the spots on the fret. Figure 6(f) shows the projections of six double strands which display the same pattern as that in the center row of the macrofiber (Figs 1(c) and (2)) and in the pawcrystals in Figure 4(a) and (c). Figure 4(c) shows other patterns as well. manJ of which arise in the computed projections. These patterns correspond to different fractional axial shifts of the double strands. Figure 6 compares computed projections of electron density with electron micrographs of negatively stained specimens. The two types of images are comparable only to the extent that regions of high electron density exclude stain and regions of low electron density are occupied by stain. Regions of high electron density in hemoglobin are found in the subunits of the molecule. These subunits are compact and have hydrophobic interiors which would be expected to exclude stain effectively. The open regions in the interiors of the paracrystals and crystals should be easily accessible to stain since large channels run parallel to the double strands. Stain would therefore be expected to penetrate regions of IOIV electron density in the sptxcimen and to be excluded from regions of high electron density, thus forming a replica of the electron density to the resolution achievable using the tichniqur of ncagativcbstaining. 6. Discussion The crystallization of deoxyhemoglobin S induced by stirring occurs by pathways which differ in their kinetics and in the intermediate forms which appea,r prior to crystallization. Our investigations indicate that the pathway of crystallization depends upon pH and the ionic composition of the crystallizing milieu. A feature common to both pathways of crystallization is alignment and fusion of fibers to form intermediates which in turn fuse into large crystals. The intermediate forms may thus provide an understanding of the relationship between the fiber and crystal structure. The final crystal structure so far appears to be independent of the pathway of its formation. In the low pH pathway described in this paper the indications are that fibers fuse to form macrofibers by a mechanism yet to be elucidated. Optical diffraction of the macrofiber reveals reflections having spacings and an intensity distribution similar to those in the fiber transform. Patterns from both fibers and macrofibers display meridional reflections at an axial spacing of 32 A. We interpret this feature of the pattern as deriving from a half stagger of the hemoglobin molecules implying that both fibers and macrofibers have similar molecular packing. The common structural unit is probably the Wishner-Love double strand. Because the molecular packing is so similar, fusion of the fibers to form macrofibers may involve little rearrangement of the individual hemoglobin molecules. We have observed macrofibers with four, five and six rows across the narrow region. This variation does not seem to indicate a major structural difference I)t%\vrrxn the particles, since optical diffraction patterns obtained from all particles

1024

T. E. WELLEMR,

R. J. VASSAR

AND

R. .JOSEPHS

are quite similar. We have, therefore, come to view macrofibers as a class of structures whose members are distinguished by their &f&ring numbers of rows. The next step in the transition involves formation of thin paracrystals in which the double strands are longitudinally but not azimuthally disordered. Although we have not yet observed specimens of macrofibers fusing to form these larger structures, we believe this is a likely mechanism because crystal development always follows macrofiber development. After several hours, alignment of the double strands occurs and well-ordered crystals are formed. The appearance of micrographs of crystals and partially ordered paracrystals can be accounted for in terms of projected electron density distributions derived from the crystal structure. Projections in which the double strands are shifted along the a axis of the crystal generate patterns similar to those in the micrographs of the paracrystals. In some regions the register of the rows is such that adjacent rows connect to produce a oharacteristic pattern of parallel stacks of segments. Fully crystalline images shots these segments in a herringbone pattern having a two-dimensional arrangement indistinguishable from that of crystals obtained by other pathways, The above observations suggest that the fibers. macrofibers, paracrystals and crystals of the low pH pathway are all closely related variations of the same molecular packing scheme. The evidence indicates that the variations involve the packing of the double strands. The computed electron density projections show that the pattern of the fret arises from a projection of the double strand taken in a direction corresponding to the c axis of the crystal. The projection is 90 to 95 A in width, which is the same as the width of the rows displaying the fret. Since the fret characteristic of the paracrystal is also present in the macrofibers, longitudinal shifts of the double strands appear to play an important role in the low pH pathway of the fiber-to-crystal transition. We have considered the possibility that macrofibers can form under in vivo conditions. Initially this appeared to be a remote possibility, since macrofibers are formed in the low pH pathway. However, in the presence of Tris the transition pH of the low pH pathway is 7.1, which is well within the physiological range. The ionic composition of the interior of the red cell is neither 50 mM in Tris or phosphate. Thus our experiments do not bear directly upon the question of formation of macrofibers in vivo, but rather emphasize the necessity of carrying out studies of fiber formation under ionic conditions which correspond to those actually in the red cell in order to obtain definitive data. Moreover, since macrofibers form several hours after fibers, their it1 viva significance would depend upon whether the ionic conditions inside the red cell accelerate the transition. On the other hand, experiments in vitro are often carried over very extended times. For instance X-ray diffraction studies have been carried out on partially oriented gels and red cells stored in capillaries for periods of several years (Magdoff-Fairchild & Chiu, 1979). Macrofibers could have formed under such conditions if a drop in pH occurred?. The observation that crystals form in the red cells in such experiments is consistent with our results which show that macrofibers spontaneously convert into crystals. t The cells were deoxygenated by placing them in & humidified atmosphere of5”,, CO2 and 0~50,~Nz, A drop in pH could have been caused by CO2 absorption and by the anerobik metabolism of the red cell (Nat&on & Nat&son, 1978).

ASSEMBLIES

OF DEONY-HbS

DOUBLE

STKAPL’DS

102.5

It is interesting to note that if the crystal structure were altered only by shifting the longitudinal alignment of the double strands the space group and unit cell dimensions would not change. This raises the possibility that species of crystals may exist which diffract similarly but in which the longitudinal registration of the double strands is altered. pH and ionic species could play a role in inducing such a longitudinal shift. However, our micrographs have not yet revealed that such packing variations actually do occur. On the basis of a comparison of various crystal forms, Love rt 01. ( 1978.1979) have proposed that deoxyhemoglobin crystals are generally composed of “acid strands” below pH 6-5 and of “alkaline strands” above pH 6.5. The acid strand is related to the alkaline strand by rotation of each molecule about its molecular dyad by about 22”. Our evidence indicates that the structure of crystals grown at pH 5 to 6 is indistinguishable from that formed in stirred solutions at higher pH. The double strand of deoxyhemoglobin S may thus be an exception to the acid and alkaline rule. This is an unexpected result in view of the change in the pathway of crystallization that occurs in the neighborhood of pH 6-5. EvidentI> arly pH-dependent changes in the structure of the double strands (around pH 65) are t)oo small to influence the final crystal form even though the mechanism of c:ry&allization is affected. The increment of energy stabilizing the double strand(s) derives from the interaction of the p6 valine (ValA3(6)/32) with the edge of the hemr l)ocket of the nearby /31 subunit (Phe(85@1, Leu(88)fil). This interaction would not be expected to be pa-dependent and may well be responsible for the invariance of the crystal structure. The presence of common contacts in crystals of different deoxyhemoglobins (Love rt CL/., 1979) suggests that the interactions responsible for sickling involvt~ areas of the molecules already predisposed to intermolecular contact. The addit’ional increment of energy provided by the /36 valine contact. although small. is sufficient to stabilize lateral interactions between these areas and thereby reduce the solubility of deoxyhemoglobin S to pathologically low levels. The conformational change which occurs upon deoxygenation is required for these abnormal attractions to exert their effect, since oxygenated hemoglobin S has about the same solubility as oxygenated hemoglobin A. These attractions lead to t’he formation of double strands. The subsequent organization of the double strands into a crystalline array occurs by many pathways, but the final crystalline states are so far indistinguishable. We thank the members of the Sickle Cell Core Laboratory of The University of Chicago for collecting the blood samples. One of us (T:E.W.) has been supported by Public Health Service grant no. Fi-T32-GM 07281 (MSTP). The work has been supported in part by a National Science Foundation grant PCM78-09815 and by National Institutes of Health grants HL 22654, HL 00434, and HL lf5005.

REFERENCES Dykes. G., Crepeau, R. H. & Edelstein, S. J. (1978). Xature (Lo&on), 272, !5Of-510. Freedman, M. L., Weissman. G., Gorman, B. D. & Cunningham-Rundles, W. (1973).

Riochpm. Pharmacol. 22, 667-674.

1026

T. E. WELLEMS,

R. J. VASSAR

AND R. .JOSEPHS

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Edited

by G. A. Gilbert