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The α-helical conformation in a sequential polypeptide
J . Mol. B iol. (1965) 13, 949- 951
The IX-Helical Conformation in a Sequential Polypeptide X-Ray diffractio n patterns hav e been obt ained from a series of sy nthetic polyp ept ides contai ning ordered sequences of y-et hyl-L-glutamy l (G) and S-benzy l-L-cysteinyl (C) residues with the general formula (GzCG)y, where x = 0, 1, 2 and 3, and y ......., 70/ (x 2) (Fraser, Harrap , MacR ae, Stewart & Suzuki, 1965). Th e pat te rn obtained fr om t he polymer (GCG)y is dominated by a series of sharp ar cs which may be indexed on an axial repeat of 26·8 A. R esults from a series of photographs t aken wit h t he specimen inclined at vari ous angles t o the X-ray beam ar e collected t ogether in Fig. l(b). The strong reflections of spacing 1·49 A on the eighteenth layer line and 4·9 A on the fifth layer line indicate that the pattern is due t o an cc-helical ph ase. Thi s is confirmed by the fact that the ba se of the cell is hexagonal, with a o = 13·2 A. The distribution of inte nsity in the molecular t ransform of a helix wit h u units in t turns (u/t helix) can be calculate d (Cochran, Crick & Vand, 1952) by the summation of a series of terms containing Bessel functions I n (21TRr) in which R is a radial co-ordinate in recipro cal space, r is a radial co-ordinate in real space and n satisfies t he select ion rul e: (1) l = tn um,
+
+
where l is the layer line index and m = 0, ± 1, ± 2.. . . In an ec-helix with all residu es equivalent, u = 18, t = 5 and the permitted valu es of n ar e plotted in Fi g. l(a) as full circles. Only t erm s cont aining low-order Bessel functions make any appreciable contribution t o the molecular transform and so diffraction is confined to layer lines on which small valu es of n are perm itted. The novel feature of the results shown in Fi g. l (b ) is that appreciable intensity is observed on certain layer lines (e.g. 4 and 9) for which n is not small for an 18/5 helix. In the iX,-helical form of (GCG)y t he distribution of residues will be as shown in Fi g. 2, and if all GCG uni ts are equivalent t he diffra ction patt ern ma y be t hought of as t he amplitude addition of the transform of an 18/5 helix with - CO.CH(CH 2 - ).NH uni t s and a 6/1 helix of opposite sense with uni ts cont aining (-CH 2.COO .C2H5 , -S.CH 2 .CeH 5 and -CH 2 .COO.C2 H 5 ) . The two helices are illustrated in Fig. 2 and additional permitted values of n for the side-chain helix, obtained by setting u = 6 and t = - 1 in (1), are shown as open circles in Fig. l(a). It will be seen that t he addit ional observed reflections all corres pond t o low-order Bessel functio ns in the molecular tra nsform of t he (GCG) side-cha in helix . Although the addit ional reflectio ns can be ac count ed for in t his way , it is surp rising that a meridional reflection of spac ing 3 X l ·49 = 4·47 A corresponding to the axial rise per GCG unit is not observed. In the ab sence of complicating fa ctors, this would imply t ha t the axial project ions of the elect ron densities of the three side-chains are not very different . Alternatively, there may be specific types of distortion or disorder present, which lead to the same situation as regards the axial projection of elect ron density without disturbing the proj ections responsible for the diffr action observed on the fourth and ninth layer lines. 949
I
I
a
•
15
I
e-Helix • (GCGyH
a
0
•
a
15 0
0
•
a
• •
a
5,-
.....
Q
•
•
•
a
a
•
a
a a 0
0
I
-7-
0-.\
•
0
-4
0-2
5
• -6
10
0
0
0
- 004
a a
lOl-
0-5
a
• .....
0-6
a
0 n
I
I
I
2
4
6
o o
10
1120 21 30 hk
0
(b)
(a)
FIG. 1 (a) n,l plot (Klug, Crick & Wyckoff, 1958) showing the orders of Bessel functions (n) contributing to various layer lines for an 18/5 helix (filled circles) and 6/-1 helix (open and filled circles). (b) Reciprocal lattice diagram showing how the observed reflections in (GCG). can be indexed on a hexagonal cell with a = 13·2 A, c = 26·8 A. Relative intensities are indicated by boldness of circle. On layer lines 4 and 9, reflections are observed which cannot be accounted for by an 18/5 helix.
-----~----~----
-----~----~-
G
-' --- 8
~-'
G -_ ...
8
--
G
-'
~~
G
_~-----8----,---
~~~
8 G
-' -'
,-
FIG. 2. Radial projection (Klug et al., 1958) of the helical form of (GCG)., showing the distribution of residues on an 18/5 helix (broken line) and the distribution of the (GCG) asymmetric units on a 6/ -1 helix (full line).
LETTERS TO THE EDITOR
951
We are indebted t o D r D. L. D. Caspar for valuable discu ssion on this latter point. Division of Protein Che m istry CSIRO Wool Research Laboratories 343 R oyal Parade, Parkville N2 Melbourn e, Victoria, Au stralia
R. D. B. FRASER T. P. MACRAE F . H . C. STEWART
R eceived 22 June 1965 REFERENCES Cochran, W ., Crick , F. H. C. & Vand, V. (1952) . A cta Cryst., Camb, 5, 58!. Fraser, R . D. B., Harrap, B. S., MacRae, T . P ., Ste wa rt, F . H . C. & Su zu k i, E. (1965). J . ,M ol. B iol . in the press . K lug, A ., Crick , F. H. C. & Wyckoff, H . W . (1958). A cta C,'yst., Oamb, 9, 436.
The IX-Helical Conformation in a Sequential Polypeptide X-Ray diffractio n patterns hav e been obt ained from a series of sy nthetic polyp ept ides contai ning ordered sequences of y-et hyl-L-glutamy l (G) and S-benzy l-L-cysteinyl (C) residues with the general formula (GzCG)y, where x = 0, 1, 2 and 3, and y ......., 70/ (x 2) (Fraser, Harrap , MacR ae, Stewart & Suzuki, 1965). Th e pat te rn obtained fr om t he polymer (GCG)y is dominated by a series of sharp ar cs which may be indexed on an axial repeat of 26·8 A. R esults from a series of photographs t aken wit h t he specimen inclined at vari ous angles t o the X-ray beam ar e collected t ogether in Fig. l(b). The strong reflections of spacing 1·49 A on the eighteenth layer line and 4·9 A on the fifth layer line indicate that the pattern is due t o an cc-helical ph ase. Thi s is confirmed by the fact that the ba se of the cell is hexagonal, with a o = 13·2 A. The distribution of inte nsity in the molecular t ransform of a helix wit h u units in t turns (u/t helix) can be calculate d (Cochran, Crick & Vand, 1952) by the summation of a series of terms containing Bessel functions I n (21TRr) in which R is a radial co-ordinate in recipro cal space, r is a radial co-ordinate in real space and n satisfies t he select ion rul e: (1) l = tn um,
+
+
where l is the layer line index and m = 0, ± 1, ± 2.. . . In an ec-helix with all residu es equivalent, u = 18, t = 5 and the permitted valu es of n ar e plotted in Fi g. l(a) as full circles. Only t erm s cont aining low-order Bessel functions make any appreciable contribution t o the molecular transform and so diffraction is confined to layer lines on which small valu es of n are perm itted. The novel feature of the results shown in Fi g. l (b ) is that appreciable intensity is observed on certain layer lines (e.g. 4 and 9) for which n is not small for an 18/5 helix. In the iX,-helical form of (GCG)y t he distribution of residues will be as shown in Fi g. 2, and if all GCG uni ts are equivalent t he diffra ction patt ern ma y be t hought of as t he amplitude addition of the transform of an 18/5 helix with - CO.CH(CH 2 - ).NH uni t s and a 6/1 helix of opposite sense with uni ts cont aining (-CH 2.COO .C2H5 , -S.CH 2 .CeH 5 and -CH 2 .COO.C2 H 5 ) . The two helices are illustrated in Fig. 2 and additional permitted values of n for the side-chain helix, obtained by setting u = 6 and t = - 1 in (1), are shown as open circles in Fig. l(a). It will be seen that t he addit ional observed reflections all corres pond t o low-order Bessel functio ns in the molecular tra nsform of t he (GCG) side-cha in helix . Although the addit ional reflectio ns can be ac count ed for in t his way , it is surp rising that a meridional reflection of spac ing 3 X l ·49 = 4·47 A corresponding to the axial rise per GCG unit is not observed. In the ab sence of complicating fa ctors, this would imply t ha t the axial project ions of the elect ron densities of the three side-chains are not very different . Alternatively, there may be specific types of distortion or disorder present, which lead to the same situation as regards the axial projection of elect ron density without disturbing the proj ections responsible for the diffr action observed on the fourth and ninth layer lines. 949
I
I
a
•
15
I
e-Helix • (GCGyH
a
0
•
a
15 0
0
•
a
• •
a
5,-
.....
Q
•
•
•
a
a
•
a
a a 0
0
I
-7-
0-.\
•
0
-4
0-2
5
• -6
10
0
0
0
- 004
a a
lOl-
0-5
a
• .....
0-6
a
0 n
I
I
I
2
4
6
o o
10
1120 21 30 hk
0
(b)
(a)
FIG. 1 (a) n,l plot (Klug, Crick & Wyckoff, 1958) showing the orders of Bessel functions (n) contributing to various layer lines for an 18/5 helix (filled circles) and 6/-1 helix (open and filled circles). (b) Reciprocal lattice diagram showing how the observed reflections in (GCG). can be indexed on a hexagonal cell with a = 13·2 A, c = 26·8 A. Relative intensities are indicated by boldness of circle. On layer lines 4 and 9, reflections are observed which cannot be accounted for by an 18/5 helix.
-----~----~----
-----~----~-
G
-' --- 8
~-'
G -_ ...
8
--
G
-'
~~
G
_~-----8----,---
~~~
8 G
-' -'
,-
FIG. 2. Radial projection (Klug et al., 1958) of the helical form of (GCG)., showing the distribution of residues on an 18/5 helix (broken line) and the distribution of the (GCG) asymmetric units on a 6/ -1 helix (full line).
LETTERS TO THE EDITOR
951
We are indebted t o D r D. L. D. Caspar for valuable discu ssion on this latter point. Division of Protein Che m istry CSIRO Wool Research Laboratories 343 R oyal Parade, Parkville N2 Melbourn e, Victoria, Au stralia
R. D. B. FRASER T. P. MACRAE F . H . C. STEWART
R eceived 22 June 1965 REFERENCES Cochran, W ., Crick , F. H. C. & Vand, V. (1952) . A cta Cryst., Camb, 5, 58!. Fraser, R . D. B., Harrap, B. S., MacRae, T . P ., Ste wa rt, F . H . C. & Su zu k i, E. (1965). J . ,M ol. B iol . in the press . K lug, A ., Crick , F. H. C. & Wyckoff, H . W . (1958). A cta C,'yst., Oamb, 9, 436.