J. Mol. Biol. (1969) 42, 379-383
The detailed structures of the various polymorphic forms of amylose (the linear polymer of CG1,4-linked D-ghCOSe) as they occur both naturally in starch and after separation, have aroused considerable interest over the years. Depending on the source, starch itself is found in three different polymorphic forms known as A, B and C starches (Katz & Van Itallie, 1930), each of which has a characteristic X-ray diffraction pattern. In addition, starch precipitated from solution takes up the socalled V-structure (Rundle & Edwards, 1943), which also exists complexed with iodine, alcohols, dimethylsulfoxide, etc. and as hydrates of a number of these forms. Al.1 these structures are also formed by “purified” amylose. We wish to report here on work now in progress on the B-form. This form has received little attention in comparison to the more tractable V-structure, but is perhaps the more interesting, as it is a naturally occurring polymorph. Various structures have been proposed for B-amylose. Rundle, Daasch & French (1944), working with starch powder patterns, indexed the reflections according to a rectangular unit cell with dimensions a = 16.0 A, b = 10.6 A (fiber repeat) and c = 9.1 A, and there has been little dispute over this cell since then. Kreger (1951) obtained “fiber diagrams” of the B-form using a microbeam method on single granules of phajus starch and proposed a structure where the unit cell contained three helica,l chains, each with three residues per turn. This structure was criticized on density grounds by Spark (1952), who proposed instead that the cell contained two 4-fold helices. Rao & Sundararajan (1968) examined all the chain conformations which had been proposed by minimum potential energy conformation analysis and considered that of these, a right-handed helix with -four residues per turn was the most likely. In the present work we are re-examining the structure of B-amylose, working with data from X-ray diffraction diagrams of oriented fibers. Potato amylose (degree of polymerization N 4500) is acet’ylated according to the method of Jeanes & Jones (1952) and oriented specimens of the triacetate are prepared as described by Sarko & Marchessault (1967). These are deacetylated following the procedure of Srnti & Witnauer (1948) to obtain oriented B-amylose specimens. The X-ray diffraction pattern of this material in helium at 98% relative humidity is shown in Plate I(a). The observed d-spacings can be indexed by a rectangular unit cell with dimensions a = 15.9 A, b = 9.1 A and c = 10.4 A (fiber repeat). This unit cell is too small t.o accommodate a chain of the type proposed below and the most likely true cell is one with the b dimension doubled to 18.2 A, while it is possible that a doubling of a adsooccurs. At the same time, a hexagonal unit cell may not be ruled out in view of the a to b ratio of 1.75 : 1 which is not significantly different from 2/3 : 1. Likewise, the space group has as yet not been established, but the only observed meridional reflection (001) is on the sixth layer line (see Plate I(b)) suggesting that the individual chains have a 6-fold screw axis. All of the reflections lie on well-defined layer lines and there is every reason to believe that the structure consists of helices with an integral number of residues per turn. Our experience with this material, like that of previous workers on starch, is that the crystallinity is greatest at high humidity, and 379
it is expected that the unit cell will contain a number of water molecules. The measured density for this material is 1.50 g cmm3, which would be in accord with a hydrated structure consisting of six residues/unit cell plus three or four water molecules/residue. This corresponds to ‘
FIU. 1. Diagrammatic representation of the maltose residue (with O(6) and O(W) omitted) showing the numbering of the atoms and the angles of rotation 4 and I# for the two residues. The residues are in the (4, I/) = (0”, 0’) position as defined by Rae et al. (1967).
in 10” increments. The ($,#) = (0,O) starting position corresponded to C(l), O(l), O(4) and C(4’), O(1’) all lying in the same plane (cf. Fig. 1). The energy due to van der Waals forces between the non-bonded atoms was calculated using the LennardJones potential functions with the constants as given by Scott & Scheraga (1966). An approximate hydrogen-bonding term of the form V,, = c/r3 (where c = -55 kcal. A3 and r is the O-O interatomic distance) was included for all interactions between atoms O(2), O(3) and O(5). The total energy for such interactions was then
A number of possible C(l)-O(l)-C(4’) bridge angles were considered, ranging from 130” to 100”. The ($,#) energy map for a bridge angle of 117” is shown in Figure 2, with superposed contours of h (the repeat distance in A per residue along the helix axis) and n (the number of residues per turn). The following conclusions are drawn. (a) For bridge angles near this value (the most likely from a study of di- and oligosaccharides (Sundaralingam, 1968)) helices with more than six residues per turn are not possible for strictly geometrical reasons. A helix with seven residues per turn can have a maximum bridge angle of 112”, and under such conditions the (+,#) map shows that the 7-fold conformations have high energy and are therefore unlikely.
4 (angle - degree) lEIG. 2. Energy contour map for & maltose residue with a bridge angle of 117”. Heavy line, pot#ential energy in kcal./maltose residue. Thin lines, contours of constant n, the number of residues per turn of the helix. Dashed lines, contours of constant h, the rise per residue in A units parallel to the axis of the helix. Positive values of h indicate a right-hand helix, negative h a left-hand helix.
(b) For helices with six or less residues per turn, 6-fold helices (both left- and righthanded) are st,rongly favored on energy considerations. This is not apparent from previous calculations which considered only non-bonded interactions (Rao et al,, 1967). These helices are characterized by hydrogen bonds between O(2) and 0(3’) of length close to 2.8 b, and fall in the range: -1.8 A < h < +1-S 8. The left-handed helix is slightly favored over the right-handed by about O-5 kcal./residue. The observed fiber repeat for B-amylose corresponds to h = kl.73 .& and that for V-amylose to h := &la33 d (Zobel, Fr ench $ Hinkle, 1967). A 5-fold helix is possible over a small range, but is less favored than the 6-fold. (c) All other helices corresponding to the observed repeat are unlikely. A small angular range allows the 4-fold left-handed helix, although this contains neither the 4-fold helix with a fiber repeat of 10.4 B nor the 4-fold helix with a repeat of 16.1 A proposed for KBr amylose (Jackobs, Bumb $ Zaslow, 1968). (The high ionic forces in the latter structure might be expected to stabilize this elongated chain.) The only possible chain conformation possessing a 6-fold screw axis is a single chain with six residues per turn. It is not possible to intertwine two 6-fold strands to retain the rise per residue of 1.73 A. Two parallel 6-fold chains could be coiled together 180’ out-of-phase as a double helix, but with each chain repeating aRer 20.8 A. This conformation predicts a third layer line meridional at 3.46 A which is not
observed. Potential energy calculations indicate that this structure is possible hut unlikely in comparison with the 6-fold single chains. Multiple helices such as two parallel out-of-phase 12-fold helices where each chain repeats after 20.8 .& are ruled out by point (a) above. Thus, the most likely chain conformation is a left-handed single helix of six residues. Although the right-handed helix cannot be excluded from consideration at this stage, the evidence is in favor of the left-handed conformation a,lso on the grounds that B-amylose is formed from amylose triacetate which has this chirality (Sarko & Marchessault, 1967). It has been shown that the chains for the V-form are helices with six residues per turn and a period of approximately 8.0 A (Zobel et al., 1967). Rao et al. (1967) have previously shown that energetically the left-handed V-amylose helix is the more stable conformation, based on non-bonded potent,ial energies alone. They proposed an 0(6)-H-0(5’) hydrogen bond between adjacent residues rather than the O(2)-0(3’) bond resulting from the calculations reported here. Our own work on V-amylose also shows that the most likely conformation is a left-handed helix and that both left- and right-handed helices are further stabilized by 0(6)-H-O(2) bonds between residues in successive turns (cf. Fig. 3). It has been
(a) FIG. 3. Cylindrical B-amylose.
of the proposed
for (a) V-amylose
established (Senti & Witnauer, 1948) that the conversion of V- to B-amylose takes place on humidification. We are proposing here that on humidification the V-form is converted into the B-form by breakage of these hydrogen bonds and that O(6)-H-(H,O)-O(2) bonding is formed which stabilizes the helix at a repeat of approximately 10.4 1(1.A cylindrical projection of the proposed conformation is shown in Figure 3. The ease with which this transformation proceeds also constitutes indirect evidence for the same chirality in the V- and B-amyloses. The calculated Fourier transform for this single chain shows fairly good agreement with the observed X-ray diffraction pattern of B-amylose considering that only one water molecule per residue has been incorporated so far. The additional waters can be accommodated with ease either in the center of the chains or between the chains when packed together.
We a#reat present working on the arrangement of the water molecules and the packing of the chains, details of which will be published in due course. We thank Dr F. R. Dintzis of the U.S. Department of Agriculture, Peoria, Illinois, for supplying the sample of potato amylose. This work was supported by Agricultural Research Service, U.S. Department of Agriculture, grant no. 1%l&100-9173(71), administered by the Northern Utilization Research and Development Division; and by the Research Foundation of State University of New York, grant no. JA-67-10-006. Department of Chemistry State University College of Forestry Syracuse, N.Y. 13210, U.S.A.
J. BLACKWELL A. SARKO R. H. MARCHESSAULTt
Received 14 January 1969, and in revised form 28 March 1969 REFERENCES Jackobs, J. J., Bumb, R. R. & Zaslow, B. (1968). BiopoZymers, 6, 1959. Jeanes, A. & Jones, R. W. (1962). J. Amer. Chem. Sot. 74, 6116. Katz, J. R. & Van Itallie, T. B. (1930). 2. physik Chem. A150, 90. Kreger, D. R. (1951). B&him. biophys. AC& 6, 406. Rao, V. S. R. & Sundararajan, P. R., (1968). Solution Properties of Natural Polymers, p. 173, Special Publ. no. 23. London: The Chemical Society. Rao, V. S. R., Sundararajan, P. R., Ramakrishnan, C. & Ramachandran, G. N. (1967). In Conformation of Biopolymers, ed. by G. N. Ramachandran, vol. 2, p. 721. London: Academic Press. Rundle, R. E., Daasch, L. & French, D. (1944). J. Amer. Chem. Sot. 66, 130. Rundle, R. E. & Edwards, F. C. (1943). J. Amer. Chem. Sot. 65, 2200. Sarko, A. & Marchessault, R. H. (1967). J. Amer. Chem. Sot. 89, 6454. Schierbaum, F. (1960). Starke, 12, 237. Scot’t, R. A. & Scheraga, H. A. (1966). J. Chem. Phys. 45, 2091. Senti, F. R. & Witnauer, L. P. (1948). J. Amer. Chem. Sot. 70, 1438. Spark, 1;. C. (1952). B&him. biophys. Actu, 8, 101. Sundaralingam, M. (1968). Biopolymers, 6, 189. Zobol, II. F., French, A. D. & Hinkle, M. E. (1967). BiopoZymers, 5, 837.
7 Present address: Department of Chemistry, University
of Montreal, Montreal, P.Q., Canada.