The structure of a paracrystalline phase of poly-γ-benzyl-l -glutamate in dimethylformamide

.I. Mol. Biol. (1967) 25, 1-13

The Structure of a Paracrystalline Phase of Poly-y-Benzyl-L-glutamate in Dimethylformamide 1). A. D.

I’ARRYt

AND

-4.

ELLIOTT

Department of Biophysics lJr&lersity of London Kilzg’s College 86-29 Drury LAne, London, lV.C.3, England (Received 11 August 1966) ‘rho X-ray diffraction patterns of oriented solutions of poly-y-benzyl-L-glutamate in dimethylformamide have been photographed at concentrations of about 501,) polymer (by weight) and upwards. The diffraction patterns have a strong nearequatorial reflection or streak and some other features usually associated with n coiled coil. They also show, however, the strong 1.50 A meridional reflection characteristic of the straight a-helical form. An arrangement in which thtl henzene rings at the ends of the side chains associate to form quasi-helici~l structures is proposed to account for the features of the diffraction pttnrn which resemble those of a coiled coil.

1. Introduction (Pauling $ Corey, 1953; Crick, 1953) as the form of the polypeptide chain in the a-proteins kerat’in, myosin, etc. The X-ray diffraction effects produced by this form are chiefly layer line a meridional reflection of spacing close to 5.15 A and a near-equatorial corresponding to about 70 to 90 A. Although the X-ray diffraction patterns of a number of synthetic polypeptides have been observed, these reflections have not been seen with samples in the solid form. Liquid crystalline solutions of poly-ybenzyl-L-glutamate in dimethylformamide and in pyridine have been described by Luzzati, Cesari, Spach, Masson & Vincent (1961) and they have suggested that, coiled coils might be present in these solutions. Sharp reflections were observed in the concentration range 15 to 55% w/w. The unit of packing is hexagonal (a = 35 A) and contains three chains. Some central diffuse scattering is observed, but this fades out at, the higher concentration, when only sharp reflections are seen. At still higher concent,rations, the reflections become diffuse and some continuous scatter is again observed. Wr have earlier reported that in this, the “complex” phase, oriented specimens show a meridional or near-meridional reflection of spacing about 5.15 d (Parry & Elliott, 1965), so giving some support to the suggestion that the structure is a coiled coil. In the course of the work now reported, this reflection has been resolved into a The “coiled-coil” has been widely

structure accepted

t Present address: Division Victorin, Australicb. 1

based on the a-helix

of Protein

Chemistry, 1

C.S.I.R.O.,

343 Royal

Parade, Parkville

NL’.

”

meridional diffraction

D. A. D. PARRY

and a near-meridional pattern has convinced

AND

A. ELLIOTT

component. A more extensive examination of the us that coiled coils are not present in these solutions.

2. Experimental

Procedure

K-Ray diffraction photographs have been taken with a camera in which the source (a Hilger & Watts semi-microfocus tube) is imaged by means of a gold mirror of toroidal shape (Elliott, 1965). This gives a beam from which the short-wave white radiation has heen removed (by the agency of the mirror, the glancing angle of which is chosen to give maximum intensity for CuKcr radiation) and which is focused to a point, so that oriented specimens may be used to produce fibre-type diffraction patterns. It is very desirable to use near-monochromatic radiation with solutions, in order to reduce the amount of radiation scattered by the solvent. The poly-y-benzyl-L-glutamate used was obtained from Koch-Light Laboratories Ltd. and has a reputed molecular weight of 320,000. With a concentration of about 50:‘; by weight of the polymer in dimethylformamide, sharp reflections are obtained. Some orientation is obtained by the use of a rotating cell described earlier but the polymer is very viscous in this phase and the orientation is poor. Better orientation of the complex phase is obtained by drawing fibres from solutions of PBLGt in dioxan, annealing in. vac1lo at 120°C to remove the solvent, and putting the flbres in a Pantak capillary tube containing a small quantity of dimethylformamide. After some days, t)he solvent is assumed to be uniformly distributed throughout the polymer. With this method it is possiblo to work \rith higher concentrations than when ilsing the rotating cell.

3. Results Diffraction photographs of oriented PBLG at successively higher concentrations in dimethylformamide are shown in Plate I(a), (b) and (c). The equatorial reflections of the complex phase may be seen in (a); the first (30 8) is lost in what appears as an equatorial streak, but which probably consists of diffuse reflections. In all three photographs a near-equatorial streak appears. The two closely-spaced arcs on the meridian correspond to spacings 10.3 and 9.6 A, respectively; the outer one is resolved into two reflections in (b), one on either side of the meridian, particularly on the upper half of the photograph. A much stronger reflection (actually a pair) at about 5 A apparently on the meridian is also seen in (a), (b) and (c). In (d) the 1.50 A reflection associated with the residue translation of an u-helix is shown; another meridional reflection (2.06 A) appears near the edge of the print but is difficult to see hecause of the background. There is also a weak reflection of spacing 1.47 A which appears to be on the meridian. Over a range of polymer concentrations the near-equatorial streak has a constant layer-line spacing of 55 A. At very high polymer concentration, when the structure is apparently breaking up, this spacing increases up to about 70 A. The observed diffraction pattern is shown in Fig. 1 and the spacings are given in Table 1. With the exception of the meridional reflection 5.06 A, the pattern can be fitted to a c-axis repeat of 165 A. If this reflection is truly meridional (which is difficult to ascertain) t’he repeat length might be 330 A. Figure 1 is made up from data recorded with different polymer concentrations, but the structure appears to be essentially the same, with very small changes in the meridional spacings over the concentration range approx. 55 to 80% (g polymer/100 g solution). The lowest concentration which was found to give a sufficient degree of orientation produced some broadening of the equatorial reflections, which at still lower concentrations are sharp. 7 Abbreviation usncl: PBLG, I’oly-y-benzyl-1,.glutamRte.

__C.“.

(b) PLATE 11. (a) Projectiorl of 6 helical arrarlgemeuts of holes. reprfwetlt.itlg hfv17rellF:rings. __- 330 .!I, diam. = 23 A, 64 holes in one pitch length. (b) Optical diffraction pat,trrrr of’ (a). Vertical lines represnrlt~ orders of 1.‘i .A.

Pitcxh

O-6-1

0 R*&') FIG. 1. Reciprocal space rotatiwt diagram for the complex plmse of PULG in dimethylformaInide. The full lines indicat,e measurements made at about, 630’,O polymer cwtlret~tration; the b~okeu lines indicate reflections observed at higher conceutrations with better molecular oriontatjioll.

4. Discussion (a) Nature

of the structure with X-ray diffractim near-equatorial streak

pattern codaireing

a

As far as we are aware, a near-equatorial reflection or streak such as is shown in Plate I has not been observed with synthetic polypeptides, though it is a well-known feature of the diffraction pattern of paramyosin (Cohen & Holmes, 1963) and of a-kcratin (Fraser & MacRae, 1961). Rudall (1965) has observed a similar streak wit,11 honey-bee silk. The near-equatorial diffraction is typical of a helical arrangement of scattering centres, for besides the strong streak (l/z* = 55 .&), weaker diffraction is seen at proportionally greater values of R* and z *. The locus of maximum intensity makes an angle of about 12” with the equator. This indicates that the diffraction comes from a helix of 1%strands of radius r whose pitch angle is about 12”, but does not, allow us to determine n or r unambiguously. The length of the repeat of pattern

4

1). A. L). I’ARL{Y

Ar;D

TABLE

Bejlections At concentration

.\.

ELLIOTII:

1

in complex phase

gtiuing shurp rejlectiom

(rrpp~oz. 5.57; w/a) :----

33, 18.8, 15.86 A (on equator) 55 2 2 A (layar limo sparing of I~~wr-vquat,oritil 10.3 :I. 1 A 8. (moritlio~~al) 9.6 d 111.(near-meridional) 5.06 A T.S. (probably mrridiouul) 5.18 A s. (probably IlecEr-lnoritliollltl) I.50 2i s. (moridioual). rldditional 3.40 4.70 2.57 1.43 2.07 1.51 1.47

d A A A ip A A

strwk)

re$ections at higher concentruthls w. meridional or near-meridional w. meridional or near-meridional m. meridional or near-meridional w. meridional or near-meridional m. meridional or near-meridional s. meridional or near-meridional m. meridional or near-meridional

w., Weak; m., modium;

s., st,rong; vs., very strong.

determined from the layer lines is approx. 165 B (or a multiple), and so the layer line giving the strong near-equatorial streak must be the third (or a multiple). The requirements of the diffraction pattern at first sight suggest a three-strand coiled coil. In a coiled coil the u-helix axis is bent into a long period (“major”) helix. The major helix has a pitch of 186 d in which there are 126 residues, which in the uncoiled a-helix would have occupied a length of 189 A. The coiled coil may bc thought of as a simple helix in which the asymmetric unit is a group of seven peptide residues. The unit translation along the major helix axis is then exactly seven times the average residue translation along the same axis. The actual repeat length of the coiled coil may depart considerably from 186 d, with very small changes in the atomic positions within one unit, but the relation just stated holds good. The mean residue translation and unit axial translation must of course be integral fractions of the period. If r0 is the radius and a the pitch angle of the major helix, the translations are, respectively, I.5 A cos a and 10.5 d cos cc,and the meridional reflections are all orders of 10.5 A cos CC.In the solid form of PBLG, the closest observed chain separation is 14.04 B (Bamford, Elliott & Hanby, 1956). If this is maintained in a t’hree-strand rope, r0 is about 8 A and u is nearly 15” for a coiled coil with an axial repeat of 186 11. If, however, we take the observed repeat 165 A, a becomes 17.5”. The spacing corresponding to the average residue translation in these two cases would be, respectively, l-45 and 1.43 A. No reflections of these spacings are seen; as stated above, the strong meridional reflection in all the forms of PBLG hitherto examined (in the solid and in solution) lies between l-495 and l-510 d. The 1.5 A reflection which we have observed in the complex phase is strong (see Plate I(d)) and could not possibly have come from any minor phase present (of whose presence there are no indications). In addition, the observed ratio between the 10.3 and 1.50 A reflection is 6.866, not 7-00 as expected. Because of these discrepancies we believe that the molecules in the complex phase of PBLG are not coiled coils, but straight helices.

PARACRYSTALLINE

,i

POLY-BENZYL-GLUTAMATE

If a coiled coil is excluded, we might next consider a helix with three turns in a repeat of 165 .&, which would give diffraction on the thirdlayer line at 55 -4, contributed by a first-order Bessel function. The pitch angle corresponding to the direct.ion of the locus of maximum intensity is about W, and this would require a radius of the helix r0 = 2.7 d. It is inconceivable that the structure of PBLG could contain such :I helix. A single helix with a pitch length of 165 ,& would not account for the observccl diffraction pattern, for it would give strong diffraction on the first and second lnyycr lines, of lvhich there is no trace; but a suitable multi-strand helix would account for the diffraction pattern. If there are n strands of pitch P, each having an n-fold screw axis, the length after which the structure repeats is P/n and the layer lines arc orders of this length. If there is not an exact n-fold screw axis all orders of I’ may appear. but those which are orders of P/,n will, in general, be stronger than their neighbours. especially in the lower orders. This seems to be the case in the complex phase of PBLG. With P = 165 x, /L = 3 all layer lines \lill be orders of 165 ii, but the third layer line, corresponding to 165/3 = 55 A% will be very strong. Similarly with I’ = 330 & n = 6, etc. the sixth order, again corresponding to 55,& will be strong. and so on. The dir&ion of the locus of maximum diflraction intensity corresponds to a poiiit on t,he 55x layer line at a radius l/R* 1. 12 A\. If this layer line is thethird, the maximum iJitennit,y in the third-order Bessel fimction would occur at this positioii if the helix radius were 8 b. If the layer line is the sixth, the helix radius required would be 14.3 B (the dominant Bessel function would now be of sixt,h order). ‘Chr~ way in which helices with these characteristics might appear in an cc-helical structurr by distortion is considered in the next sub-section. (b) Cha9age.sin helix ~ymmety

produced by distortions

in a?&a-helix

‘l’hc lower part of Pig. 2 shows the helix net or radial projection of an x-helix with 1S residues in five turns, with points showing the positions of the atoms of one species. Evidentlyr, the structure may be represented by one helix of five turns in 27 .& (broken lines) or by three helices each of one turn in 27 A4 (chain lines) or by seven helices each of one turn in 7 x 27 = 189 8, etc. So long a,s all the units on the helices are equivalent, the repeat length remains 27 A and so no indication of the presence of a helix with a longer repeat appears. If, however, (either because of steric distortions or because of the presence of different, chemical units) the units are not equivalent, it is possible for periodicitics other than that of the single, basic helix to appear. Two examples of this may be given. In poly-L-alanine, an examination of the packing has shown that, because of comacts between the methyl groups, three kinds of environment occur, and the asymmetric unit contains three residues (Elliott & Malcolm, 1959). In this case the heliccs shown by the chain line in the lower part of Fig. 2 are no longer equivalent, and t)hcy each contribute one residue t,o the asymmetric unit. For these helices, therefore, a meridional reflection corresponding to the residue translation 2716 = 4.50 -4 should a,ppear, with higher orders. In poly-L-alanine the situation is not quite as shown in Fig. 2, because the helices are not of the 18/5 but of the 47/13 type, with a c-axis repeat length of 71 $. For this helix, the distortions which destroy the equivalence of the helices shown by chain lines do not affect every residue to the same extent. That part of the Fourier transform of the molecule which is caused by the distortions is therefore broadened along the &axis and has sufficient magnitude

D. A. D. PAItltY

AND

A.

ELLIOTT 189 8,

108

81

27

0 0

360”

lb. 2. Net diagram for an cc-helix with 18 residues and 5 turns in a repeat length of 37.0 if. The upper part shows portions of a helix with a longer repeat length formed by attractions between side chains (specifically, between benzene rings).

on the 16th layer line to give a reflection, corresponding to 4.3 A, as observed. Similar distortions from packing occur also in poly-y-methyl-n-glutamate. In one specimen the helix was found to be of the 18/5 type, with a c axis repeat length of 27 A (Brown, personal communication) and here, as would be expected, a meridional reflection 4.50 A was observed. The effect of chemical differences between residues is shown very strikingly in a recent paper (Fraser, MacRae & Stewart, 1965). A polypeptide was made by polymerising the tripeptide GCG, where G is a y-ethyl-L-glutamyl and C an S-benzyl-Lcysteinyl residue. This polymer forms u-helices in which the C residues are wholly in one of the three helices shown by chain lines in Fig. 2. Layer lines allowed by the selection rule for helices with six residues in one turn were observed in the diffraction pattern of the polymer, in addition to those of the 18/5 helix. It is to be expected that in some circumstances the diffraction pattern associated with what may be called the 189 A or long-period helices of Fig. 2, shown by full lines, may appear. The long-period helix has much in common with a coiled coil. In fact, the formation of a coiled coil is one way of distorting the seven long-period helices so that they are no longer equivalent. The meridional reflections for the long-period

PARACRYSTALLINE

POLY-BENZYL-GLUTAMATE

7

helices are orders of 105 (= 7 x 1.5) A whereas for the coiled coil they are orders of 105 cos M.(Crick, 1953). (c) Nodels for the complex phase in poly-y-benzyl-~-glutamate The near-equatorial streak on the 55 A layer line in Plate I and the 10.3 A meridional reflection are the most notable indications of a departure from the symmetry of an u-helix. The distortions required to produce such effects are considerable, and are naturally ascribed to interactions between the benzene rings of PBLG. In the homologous polymer poly-j3-benzyl-L-aspartate (Bradbury et al., 1962), the benzene rings which terminate the side chains associate to form stacks, and the st,ructure is distorted from an u-helix to the topologically similar w-helix. This has a 4-fold screw axis and every fourth ring belongs to one of the four stacks, which in consequence are parallel to the helix axis. The energy of interaction is appreciable, for the NH.. .O peptide hydrogen bonds are decidedly not linear. The distance between interacting rings is 5.3 A. Since the benzene rings have a van der Waals thickness 3.4 A perpendicular to the plane of the ring, they can come into contact only by tilting so that their normals are inclined at about 50” to t’he helix axis, and this they appear to do. The benzene rings of the longer side chains of PBLG do not associate to produce stacks parallel to the helix a,xis (no w-form has been observed). Probably because of their greater length, a somewhat different arrangement is possible which allows many benzene rings to associate without distorting the u-helix of the peptide groups. The lower part of Fig. 2 shows the helix net for an 18/5 u-helical arrangement of points (representing benzene rings) on a radius of 8 A. The unit of repeat is taken as 27 A; this is nearly half the observed layer line spacing 55 A. The repeat length is perhaps altered from the usual value 27 A, but the difference between the observed repeat 165 A and the length of six repeats of a normal u-helix is within the experimental error and is not significant. The middle part shows a selection of these points, namely those on two adjacent long-period helices within a 54 A length of the u-helix (two repeat lengths), and the upper part shows another similar pair 54 A further up. Because the side chains carrying the terminal groups are long, movement of the groups in the direction of the arrows can result in each pair of long-period helices forming a single stack which is inclined to the helix axis, such stacks being stabilized by attraction between the benzene rings. Figure 5 shows how, by tilting the benzene rings so that their normals make an angle of about 49” with the direction of the stack, adjacent rings may be brought into contact. It will be noted that the structures hitherto described are formed on one molecule. Since there are seve?t undistorted long-period helices on each molecule, it is evident that an isolated molecule could form only three stacks along its whole length by pairing, leaving one long-period helix with its benzene rings unsatisfied. A structure containing such molecules might give a near-equatorial streak as observed, but all the meridional reflections would be orders of 10.5 A, whereas we have observed a strong meridional reflection 10.3 A (a,nd some orders). The difference, though small, is significant and its consequences are interesting. The average separation between successive units in the inclined stack in Fig. 2 is 10.5 A/ (2 cos u), which for u = 15” is 5.44 A. This is appreciably greater than in the w-form. Evidently if some of the unpaired rings (one-seventh of the total) could be included within the stacks, the average distance between rings could be reduced.

D. A. D. PARRY

AND

.\.

ELLIOTT

FIG. 3. The filled black circles indicate a modification of the packing arrangement proposed by Luzzati et al. for the complex phase in PBLG (the circle indicates the diameter of the /I-carbon atom). In addition, projections of quasi-helices formed by interacting benzene rings are shown within a triad and within a hexad of molecules.

between molecules This is not possible within a single molecule, but a give-and-take could lead to a more favourable energy balance. That some linking of molecules occurs is suggested by the fact that the intermolecular distance remains constant over a wide concentration range (about 15 to 55%: Luzzati et al., 1961). Over this range, the molecules pack on a hexagonal net of side 35 A with three molecules in each unit. The number of chains passing through each unit is deduced from the polymer concentration and the size of the unit; it is unlikely that this could be in error and that the number should be two or four. Although the X-ray diffraction data do not demand it, it is permissible to suppose that all molecules lie on the points of a of vaca,nt sites. The molehexagonal net of side 175 A, with a regular arrangement cules are shown by filled circles in Fig. 3. The vacant site, surrounded by six chains, is a likely place for intermolecular interactions between side chains. Provided that they are of short length, a number of sta,cks of interacting benzene rings can be formed along the whole length of a molecule, corresponding points in each stack being displaced 54 A along the helix axis as shown in Fig. 2. If each stack is made to contain about ten or eleven rings, and is formed on the side of its parent helix facing the vacant site, the lower end of each stack on, say, molecule A can be connected to the upper end of a stack on the neighbouring molecule B. The lower ends of the stacks on C can similarly be connected to the upper ends of the stacks on A. In this way, six strands of interacting benzene rings can be made to surround t,he vaca,nt site. These strands are shown in Pig. 4, where they are drawn as if they form helices of pitch 6 x 55 = 330 A, though in all probability they are not perfectly regular. The net is drawn as if seen from the position of the vacant site, and the end benzene rings of the st,acks contributed by molecules A, B and C are indicated. If each short strand preserved the symmetry of its parent helix (to which its benzene rings are connected by side chains) the whole six-stranded structure would, in projection, appear to consist of six short circular arcs connected as shown at D in Fig. 3 to form a cusped figure, It is likely that the shapes achieved will lie between

P.4R.4CRYSTATdLIKE

POLT-RESZ7-L-C,T,UT.4JIA’I’F,

!I

‘l’l~e this extremr form and a regular helix. They ma,y be termed quasi-helices. quasi-helices would be expected to give a diffract,ion pattern with a strong layer line rorrcsponding to about 55 A. We have assumed that the slightly diffuse X-ra,v diffraction patterns which we have observed with oriented specimens at concentrations ,just above the maximum for sharp reflections correspond to a slightly disordered form of the complex phase. The orientation which we have been able to induce in tllc l)liasr giving sharp reflections is not sufficient to show all the details of the diffract ion pattern. The stereochemistry does not enable us to state precisely the number of bcnacue rhlgs within each short stack on a parent, helix, but it may be chosen in conformit>\vith the X-ray data. Meridional reflections, orders of 10.3 A, are observed (inclrtdiug IIIC seventh order 1.47 A) as well as a strong meridional reflection 1.50 A. This shows the presence of two axial periodicities. The 1.5 d reflection, associated with sc-helices, doubtless corresponds to the axial translation of the peptide groups, the 10.3 .k reflect,ion that of the side chains, or of part of them. The off-meridional “reflection” 5.15 A would, on this interpretation, probably be the peak of the layerIiuo streak aorresponding to the 5.4 A pitch of the u-helices. We suggest that the nvrrage a,xial translation of the benzene rings in the quasi-helices is lo*3 A/2. Karli unit, contains tl~‘o benzene rings, for even if the rings were equally spaced along the quasi-helices, the attached side chains would not be, since they a,re derived from t~rlo long-lleriod helices. The number of units in t’he l’itch length of a quasi-helix (330 i\) is tllcxrefore 32, and the number of benzene rings is 64. Since this is ilot divisible by six, tllc six parent helices do not contribute equally to a given strand. Four parent llelic.c~s donat,e 11 benzene rings, the others 10 each. It is because of this inequality t)hat reflections indicat,ing a repeat length of 165 i (and not 55 A) are observed. as will now be shown. The net for a quasi-helix (idealized to a perfect helix of points) is shown in Fig. -J. The axial periodicity 10.3 rather than 5.15 A is indicated by the lines representing t,hr connection to the remainder of the side chains. It n-ill be seen that, although the pitch lcngt’h is 330 A, t,he repeat length for a perfect helix would be reduced to 165 A. since the number of chains a’nd of units are bot’h divisible by two. It, is not reduced further, since t,here is no larger common divisor. There is some reason for believing t#hat the helix is not in fact perfect, but distorted and that the period may be 330 A. This is suggested by our observation of a reflection 5.06 A apparently on the mcridiou (which would he on a layer line of a 330 A, but not of a 165 a repeat’) and also 1)~ somrb observations on the complex phase of PBLG in acetophenone by Mr *J. Squiw. no\v iti progress. Figure 5 has been drawn for a quasi-helix with radius 11.5 ,A. It seems likely that. the radius will be less than this in some pa.rts of the quasi-helix. The dista.nce bet\vceu ad.jacent benzene rings depends on the radius of the quasi-helix, and is not likeI> to be grcatcr than that given in Fig. 5, namely 5.27 A. This is almost the sa.me as the separation 5.3 A of the benzene rings in the w-form of poly-/3-benzyl-L-aspartate. The shortening of the separation (5.27 instead of 5.44 a) obtained by including a few extra benzene rings no doubt stabilizes the syst’em. The number of interacting benzene rings in the repeat length of the structure described is 384 out of a total of 660, which is just over 417 (each molecule contributes to two quasi-helices). It is possible that three-strand staoks of benzene rings may be formed at places slich as TCand P. These structures would have a pitch length 165 B. It is not easy t,o

IO

n _A _-

c

c

-

c

c

c

c

t

t

e

c

c

e

*

t

-330 K

c

c

r

-275

c

c

c

c

t

c

e

c

e

o-

c

t

-220

t

9-

t

m-

t

e

- 165

-110

-55

SO

Fro. 4. The helix net for the 6 helicd stacks of benzene rings surrounding the vacant site at V in Fig. 3. The rings contributed by molecules A, B, etc. in Fig. 3 are included within the length AA, BB, etc. The structure repeats after 165 .& Note that the lengths AA, BB, etc. are not all equal.

PARACRYSTALLINE

POLY-BENZYL-GLUTAMATE

11

see, using models, whether there is room for such quasi-helices. In any case, there arc not sufficient side chains to fill all the sites of 6-fold and 3-fold quasi-helices, but it, may be that further stabilization is achieved by some stack formation at such places. We have not been able to assign a specific role to the solvent in inducing the peculiar structure of the complex phase.

c-O\ H2

5. The relat,ive position t of neighbouring FIG. The angle between the stack and the normal rings of thickness 3.4 A into contact.

benzene rings in one of the six stacks of Fig. 4. to the ring (49”) is chosen so as to bring planal

(d) Optical diflraction patterns For the initial examination of a molecular model, optical diffraction is convenient (Lipson & Taylor, 1951), especially when the continuous transform is required for comparison with observed layer line streaks. For this purpose masks were made in which each benzene ring was represented by a single hole. Projections parallel to the helix axis were made for each of the four quasi-helices shown in Fig 3; the a-axis translation for the benzene rings was 5.15 .& as explained above. No attempt was made to introduce a 10.3 A period (by including other parts of the side chains). A reproduction of the mask and of its optical diffraction pattern is shown in Plate 11, for the arrangement of benzene rings forming six strands of perfect helices. The strong streak on the 55 d layer line extends from 1/R* ~20 A to about 8 A. The streak on Plate I(a) extends over about 15 to 10 d. The maximum in the optical pattern is at about l/R* = 11.5 A, w h ic h is near the observed one. The two model structures of three-strand helices also have diffraction patterns in which the 55 A layer line has a maximum approximately where the observed one occurs. The diffraction pattern of the 6-fold ousped arrangement does not fit the observed one, for the maximum is at

12

D. A. D. PARRY

RKD

A. ELLIOTT

l/R* = 30 8. This structure would require rather drastic modification to make it satisfactory. It is clear that either a 3-fold or a B-fold arrangement of quasi-helices can account for the near-equatorial streak. It is possible that both occur. If this is so, there may bc an element of randomness in the structure, for (as explained earlier) a molecule camlot be part of two triads as well as of two hexads.

5. Concluding

Remarks

The observed diffraction pattern, according to the suggestions made, comes from an arrangement in which the peptide groups and the side chains up to the p-carbon at’om at least are in u-helices. The benzene rings at the ends of the side chains (or most of them) have a different arrangement, and form quasi-helices. These probably receive contributions from six molecules. The x-helical part of the molecule will diffract X-rays to give a pattern in which the most prominent features are layer ines with spacings 1.5 b and 5.4 8. In solid PBLG, the maximum intensity on the 5.4 A layer line corresponds to a spacing of 5.25 A (Elliott, Fraser $ MacRae, 1965). If the outer part of the side chains has not the symmetry of the u-helix (as in the complex form) the position of maximum intensity on the layer line may be expected to move outwards, and hence to correspond to a shorter spacing. The off-meridional “reflection” 5.18 A may correspond to such a point of maximum intensity. The degree of orientation is hardly sufficient to allow certainty in this. The kind of distortion proposed (caused by interaction between benzene rings) would probably affect all the side chains in one repeating unit to differing extents, and it would be difficult to take account of the diffraction by all atoms in the side chains. The arrangement of quasi-helices accounts for the observed near-equatorial reflections, and in principle, reflections near 10 A in the meridional region must occur as explained in section (c) above. The nature of the disorder which produces equatorial streaks has not been elucidated, partly because the experimental data are not yet adequate. It is not known for certain whether such a streak is present at concentrations when the equatorial reflections are sharp. The streaks observed at higher concentrations may indicate a random occurrence, on lattice sites, of different structures (such as quasi-helices of t,hree or of six strands, or perhaps of quasi-helices of differing degrees of perfection). The problem is complicated by the imperfect lattice development at higher concentrations. While the structure which we propose for the complex phase in PBLG has not been tested by comparing calculated and observed intensities (which would be very dificult) we believe that we have shown some of the effects which can be produced by side-chain organization. These include a near-equatorial streak similaito that observed in the diffraction pattern of the u-proteins (keratin, etc.). Although the nature and variety of side-chain interactions in these materials are very different from those in PBLG, the question must be asked whether sufficient attention has been paid to this aspect of their structures. We are pleased to record our indebtedness to Professor Sir John Randall for the excellent facilities provided, and to Mr A. Fasoli for much help in instrument construction. One of us (D.A.D.P.) wishes to thank the Science Research Council for a maintenance grant during the past three years.

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