STEREOCHEMICAL CRITERIA FOR

POLYPEPTIDE AND PROTEIN

CHAIN CONFORMATIONS
II. ALLOWED CONFORMATIONS FOR A PAIR OF PEPTIDE UNITS

C. RAMAKRISHNAN and G. N. RAMACHANDRAN

From the Centre of Advanced Study in Biophysics, University of Madras, Madras,
India, and the Biophysics Research Division, University of Michigan, Ann Arbor

ABSTRACT The conformation of a polypeptide or protein chain may be

specified by stating the orientations of the two linked peptide residues at each
alpha carbon atom in the chain, namely the two dihedral angles 4, 4' about the
single bonds N-aC and aC-C' from a defined standard conformation. By us-
ing certain criteria of minimum contact distances between the various atoms, the
allowed anges of (4), d/) have been worked out for three values of the angle
N-aC-C' (f), namely 105, 110, and 1150 for non-glycyl, and 110 and 115° for
glycyl residues. The theory is compared with all the available crystallographic
data (up to early 1965) on simple (di- and tri-) peptides, cyclic peptides, poly-
peptide and protein structures, and the observed data fully support the conclu-
sions from theory. The effect of the gamma carbon atom, in its three possible
positions, is also discussed, and is found to alter the outer limits of the allowed
region of (4), 4') only slightly. The paper contains exhaustive references to the
published data on these structures, using x-ray diffraction.

INTRODUCTION
The conformation of the backbone of a polypeptide or protein chain can be com-
pletely specified by giving the relative orientations of the two linked peptide groups
at each a-carbon atom. When the relative orientations of the peptide group are the
same at every a-carbon atom, then the chain takes up a regular helical folding. In
Part I (Ramakrishnan, 1964) methods were described of calculating the parameters
of such a helix, mainly the number of residues per turn, n, and the unit translation
of the residue along the helix, h, from a knowledge of the geometry of the peptide
group and the relative orientations of the linked peptide groups. However, not all
possible orientations will be stereochemically allowed, because of the short contacts
between the atoms of the adjacent residues. A study of this type has been made using
certain definite stereochemical criteria and the preliminary results corresponding to
the angle N-aC-C' (T) = 1000 have already been published (Sasisekharan, 1962;

909
Ramachandran, Ramakrishnan, and Sasisekharan, 1963a and b). This paper
describes the fuller details of the results for this value of the angle, 7, and also for
two other values of r, namely 105 and 1150. In addition, a complete survey of the
structures of di- and tripeptides, cyclic peptides, and polypeptides has also been
made to check the predicted ranges of allowed conformations. These results are
discussed in addition to a study of the conformations which occur in myoglobin.
It may be mentioned that although studies of this type have been reported so far
in the literature (Huggins, 1943; Bragg, Kendrew, and Perutz, 1950; Pauling, Corey,
and Branson, 1951; Low and Baybutt, 1952; Low and Grenville-Wells 1953;
Donohue, 1953; Lindley and Rollett, 1955; Shimanouchi and Mizushima, 1955;
Scheraga, 1960), they have not been definitive. Mizushima and Shimanouchi (1961)
have taken into account the restrictions or barriers due to the rotation about the
two single bonds meeting at the a-carbon atoms and have preferred nine conforma-
tions for the polypeptide chain. More recently, De Santis et al. (1965) have con-
sidered this problem from potential energy considerations, and Nemethy and
Scheraga (1965) have obtained interesting results regarding extended chains and
the formation of closed loops containing S-S bridges.
The notation used here for representing the relative orientation of the linked
peptide groups is the same as that described in Part I (Ramakrishnan, 1964). The
rotations of the two peptide groups are denoted by two dihedral angles (4, 4')
respectively, these being measured from an initial standard conformation 4 = 4' =
00, in which the planes of the two peptide groups lie in the plane N-aC-C` containing
the two axes of rotation, and the NH groups of the two residues are pointing towards
each other.'
DETERMINATION OF ALLOWED CONFORMATIONS
In order to determine the allowed conformations, the contact distances between the
atoms in the adjacent residues have to be examined using criteria for minimum van
der Waals contact distances. This is best done by first working out the positions of

lAt a recent conference of some of the representative workers in this field held in Bethesda,
it was decided to denote the two dihedral angles about the bonds N-Ca and C.-C' by 0
and 4' respectively, the sense of rotation being the same as that adopted here. The fully extended
chain, with N-H and C'=O trans with respect to one another, is to be taken as the standard
conformation with 0 = * = 0.
It is readily seen that the new 0 is the same as the old 0, but that 4' = 1800 + .' (and
o' = 180° + 4'). All the data reported here are thus readily converted into the (O, 4') coordi-
nates. The diagrams in Figs. 2, 3, and 6 have to be shifted up by 1800, or half the total length,
along the vertical direction. In particular, the right- and left-handed alpha helices will have (¢,
4') equal to (1330, 1230) and (2270, 2370). As before, a helix with (-p, -4') will be inverse
to one with (q, 4); i.e., it will be of opposite sense, but having the same number of turns
per unit.
This paper was finalized well before this meeting, and so the older conventions are adopted
here. However, it is proposed to use the new notation in the following papers in this series.

910 BiopHysicAL JouRNAL VOLUME 5 1965

the atoms in the two residues for various values of (4), 4'). Actually, the coordinates
of the atoms in the two residues were calculated with respect to a suitably chosen
fixed coordinate system, from which the various contact distances were calculated
for the various values of (4, f'). The following fixed system of coordinates was
found to be convenient for this purpose.
The a-carbon atom C2 at which the two residues [C l-C1'O-N1H1-C2] and
[C2-C2'02-N2H2-C3J are linked together is taken to be the origin of coordinates
of a system of axes OXYZ defined as follows. The plane NlC2C2', which remains
unchanged whatever be the rotation of the two groups, is taken as the XY plane
with the Z axis upwards. The direction C1C2, which lies in the XY plane for = 0°,
is taken as the Y axis. The Z axis is the third perpendicular direction, such that the
X, Y, and Z axes form a right-handed system of coordinates. This is shown in Fig. 1.,

/ \, FIGURE 1 The two peptide groups in the initial

conformation 0 = o' = 00. The coordinate
0
°°1 axes X, Y, and Z used in the present study are
Cl also marked.

Throughout this study, the peptide group has been taken to be planar with NH
and CO groups in the trans configuration and having the dimensions given by Corey
and Pauling (1953).
If x, y, z and x', y', z' are the coordinates of an atom before and after rotation,
the relations connecting these are given by (Whittaker, 1952)
xi = (a2 + b2 _ c2-d2)x + 2(bc-ad)y + 2(bd + ac)z
y' = 2(bc + ad)x + (a2 - b2 + c2 - d2)y + 2(cd- ab)z (la)
z' = 2(bd - ac)x + 2(cd + ab)y + (a2 - b2 _ c2 + d2)z
where
a = cos (w/2), b = L sin (c/2), c = M sin (w/2), d = N sin (w/2) (lb)
m being the angle of rotation and L, M, N, the direction cosines of the axis of
rotation with respect to the chosen system of coordinates. The sign of a) is taken

RAMAKRISHNAN AND RAMACHANDRAN Polypeptide and Protein Chain Conformations 911

to be positive, when the rotation is counterclockwise viewing along the axis towards
the origin.
The angles of rotation and direction cosines of the axes of rotation corresponding
to the two peptide groups about the axes N1-C2 and C2-C2' are given in Table I.
The coordinates of the atoms C1, C1', O1, and H1 were evaluated at intervals of
100 of 4) and the coordinates of the atoms 02, N2, H2, and C3 were evaluated at
intervals of 100 for q', using the relations (1) for three values of the angle , namely,
105, 110, and 1150. In fact, all the calculations corresponding to r = 110 and 115°
were carried out using a desk calculator, while those corresponding to 7 = 1050
were carried out much later using an Elliott-803 electronic computer.
The next step towards working out the allowed conformations is to choose a
set of permitted minimum contact distances between the different types of atoms.
Only those conformations (4, 4') which do not have any of the contact distances
less than these minimum values now become allowed. Two such sets are given in
Table II, termed "normally allowed" and "outer limit" contact distances. These are
TABLE I
ANGLES OF ROTATION AND THE DIRECTION COSINES OF THE
AXES OF ROTATION USED IN THE RELATIONS (1)
Direction cosines of
the axes of rotation
Axis of Angle of
rotation rotation L M N

NI-C2 0 -sin A, -cos b6 0

C2-C2' +' -sin a,' cos a,' 0
where 5a = CiC2Ni, 6' = 180°- (r + al) and r = NlC2C2'

TABLE II
MINIMUM CONTACT DISTANCES ASSUMED
Contact Normally allowed Outer limit
A A
C ...c 3.20 3.00
C' ... C' 2.95 2.90
C ...0 2.80 2.70
C N 2.90 2.80
C ...H 2.40 2.20
O ...- O2.70 2.60
O *--.N 2.70 2.60
O *--.H 2.40 2.20
N---.N 2.70 2.60
N ... H 2.40 2.20
H *--.H 2.00 1.90

912 BIoPHYsIcAL JouRNAL VOLUME 5 1965

essentially the same as those given by Ramachandran, Ramakrishnan, and Sasisekha-
ran (1963) arrived at from a study of the contact distances observed in structures
of various organic compounds. Contact distances in between the two limits have also
been observed in actual crystals, but not as frequently as the normally allowed
values.
In order that the results can be applied to actual polypeptide and protein struc-
tures, the contact distances of the side chain atoms with those of the backbone
atoms must also be considered. However, in this study, only the contact distances
of the backbone atoms with the 8-carbon atom were considered (see Appendix for
the effect of C7).
The position of the ,8-carbon atom was fixed (using a stereographic projection
technique) in such a way that the distance Ca-Cp is 1.54 A and that the angles
N1-C2-Cp and C2'-C2-Cp had tetrahedral values (119030'). Of the two possible
positions of Cp, the one corresponding to the L-type of residues has been used
in all these studies and hence the results pertain to L-amino acid residues. However,
for 1-amino acids, the corresponding allowed conformations will be (-4, -4/), where
(4, 4f') refers to L-amino acids.
Since the positions of the atoms N1, C2' (which lie on the axis of rotation) and C,
do not change whatever the angle of rotation of the two residues is, only the follow-
ing contact distances were calculated initially: (a) from Cp and C2' to C1, C1', O1
and H1 for values of 4 from 0 to 360° at intervals of 100; and (b) from Cp and
N1, to 02, N2, H2, and C3 for values of 4' for 0 to 3600 at intervals of 10°.
These calculations were carried out corresponding to all three values of T, namely,
105, 110, and 1 150. When the ranges of 4 and 4,' allowed by each contact, using the
minimum contact distances given in Table III, are combined, the resulting ranges
given in the first half (a) of Table III are obtained.
Within the permitted ranges of 4 and 4,' given in Table III, the contact distances
between the hydrogen atom, H, attached to the a-carbon atom and the atoms of the
backbone were also examined and it was found that for all the three values of r, no
further restrictions arise due to these contact distances.
When the remaining contact distances between the atoms occurring in the back-
bones of the two residues (the positions of which change during the rotations
4 or 4,') were examined in the permitted range given in Table III, contact distances
less than the minimum allowed values were found to occur for some values of
(4, 4') and thus some more conformations became disallowed on account of these
short contacts. The resulting diagrams showing the regions of stereochemically
allowed conformations are shown in Figs. 2a, b, and c corresponding to the three
values of T. In all these figures, the normally allowed regions are shown by continuous
lines, while the boundaries of the region allowed by the outer limit values are shown
by broken lines.
In order that the results can be applied to the conformations occurring with glycyl

RAMAXRISHNAN AND RAMACHANDRAN Polypeptide and Protein Chain Conformations 913

 0
360

18C
_U I

10'~~~~~~~~~0 36

n e0
ong
50O
¢? ~~~FIGuRz 2a
O DI -AND TRIPEPTIDES - NON.GLYCYL
* DI-AND TRIPEPTIDES- GLYCYL
CYCLOHEXAGLYCYL HEMIHYDRATE
O FERRICHROME- A OINO*OLYCYL
G a-HELIX RIGHT-HANDED 3
~ ~_f aw~~
L _ __ J
__ oc-HELIX LEFT -
^ POLY-GLY- PRO-HYPRO
HANDEDJ'"3)
I*ft
me ~ O COLLAGEN
o SILK
W-FORM OF POLY-p-BENZYL-L-ASPARTATE
RIBBON STRUCTURE - 2 27- HELIX
®) (-HELIX (5117)
it-HELI X (4-416)
) 27-HELIX
30io1HELIX
4-314-HELIX
P-C- PUCKERED CHAIN
a V ANOTHER POSSIBLE TRIPLE HELIX
a

180i -I
a
a

T 'I

I I

I I

-
I i .
0O 180- 36so
FIGURE 2b
 0 180 360

FIGURE 2c
FiouRE 2 The normally allowed ( ) and the outer limit (----) regions of
(0, 0') for (a) r = 1050, (b) r = 1100, (c) r = 1150. The conformations of known
simple peptides, polypeptides, and proteins are also marked in Fig. 2b.

residues (i.e. where there is no 8-carbon atom), the contract distances between the
hydrogen atoms attached to the a-carbon atom and the various backbone atoms
were evaluated for r = 110 and 1150. The two hydrogen atoms were fixed at
tetrahedral directions to N1-C2 and C2-C2' and at a distance of l.OA from C2.
It was found that both for X = 110 and 1150, for all values of p and q', there were
no short contacts between the hydrogen atoms attached to the a-carbon atom and
the backbone atoms. As a result, the permitted ranges of c& and 4' are larger for
glycyl residues than for non-g1ycyl residues. The permitted ranges are given in the
second half (b) of Table III. The regions of allowed conformations for glycyl
residues when all the contact distances were taken into account are shown in Figs.
3a and b, where shaded regions are those allowed by only the outer limits, but not
the normally allowed contact distances.
It may be mentioned that for glycyl residues the contact distances between any

RAMAKRISHNAN AND RAMACHANDRAN Polypeptide and Protein Chain Conformations 915

 TABLE III
RANGES OF o AND q' ALLOWED BY THE CONTACT DISTANCES BETWEEN
THE ATOMS N1, C2', Cp AND THE REMAINING BACKBONE ATOMS IN
THE TWO RESIDUES FOR NON-GLYCYL RESIDUES AND BETWEEN
THE ATOMS N1, C, H2', AND THE REMAINING BACKBONE
ATOMS FOR GLYCYL RESIDUES
Normally allowed range Outer limit range
qb) 4' qb 9b'
(a) Non-glycyl residues
95 to 141' 0 to 120° 38 to 1630
105' 22 to 1150 and and and
301 to 3090 357 to 3600 289 to 3220
92to 1790 0to 1350 20to1900
1100 22 to 1270 and and and
300 to 3200 225 to 241° 289 to 3380
98 to 177° 4 to 1470 0 to 188°
1150 27 to 139° and and and
301 to 3300 213 to 2370 290 to 3600
(b):Glycyl residues
0to1270 0to1350
1100 and 40 to 3200 and 22 to 3380
233 to 3600 225 to 3600
0 to 139° 0 to 1470
1150 and 30 to 3300 and No
221 to 3600 213 to 3600 restriction

two atoms of the backbone in the adjacent residues for any conformation (4, 4') is
the same as for the inverse conformation (-cp, -4') [as can be seen from the rela-
tions (1), where, if x', y', and z' are the coordinates of a backbone atom for 4
(or 4') then the coordinates of the same atom for -4 (or -4') are x', y', and -z].
The results shown in Figs. 2 and 3 will be useful in investigating the conformation
at an a-carbon atom when the two dihedral angles 4, and 4' are known. For poly-
peptide and protein chains, the parameters that are obtainable from x-ray diffrac-
tion studies are the number of residues per turn, n, and the unit translation, h,
(Ramachandran, 1960) along the axis of the helix. Hence, the stereochemically
allowed combinations of n and h were evaluated using the detailed results of the
calculations described in Part I (Ramakrishnan, 1964) and are represented graphi-
cally in Figs. 4a, b, and c for the three values of T. In these figures also, the regions
of (n, h) allowed by the normally allowed contact distances are shown by continuous
line boundaries, while those allowed by outer limit values are shown by broken lines.
The origins in these figures correspond to n = ± 2, and h = 0 A. The positive and

916 BIoPHYSICAL JouRNAL VOLUME 5 1965

 FiouRE 3a

Ia
I~~~~~~~~~~~~~~~~~3
.i.

low 34 aO
_-

FIoUR 3b

FIOURE 3 Allowed regions of (0, 4") for (a) r = 110', (b) r = 1150, for glycyl
residues. The shaded regions represent conformations which are allowed only by the
outer limit contact distances. The glycyl conformations observed in simple peptides are
also marked in Fig. 3a: 0 = Di- and Tripeptides; 0 = Cyclic peptides; 5 =
Polyglycine II.

RAMAKRISHNAN AND RAMACHANDRAN Polypeptide and Protein Chain Conformations 917'

negative directions of the X axes correspond respectively to positive and negative
values of n, but h is always taken to be positive. In other words, plots corresponding
to all right-handed helices lie to the right of the Y axis and similarly points repre-
senting all left-handed helices lie to the left of the Y axis. In Fig. 4b, which corre-
sponds to T = 1100, the various observed and postulated polypeptide and protein
structures are also plotted.
DISCUSSION OF THE RESULTS
From Figs. 2a, b, and c, it can be seen that the general nature of the regions of
allowed conformations (4, 4)') is the same for the three values of r. An interesting
feature is that the region around (2200, 600), where the left-handed a-helix occurs,
is not allowed for T = 1050, while it is allowed for T = 110 and 1150.
In order to have a quantitative estimate of the results, the area under the allowed
regions may be expressed as a percentage of the total area of the plane -0'. These
are given in Table IV. There is an appreciable increase in the range of allowed
regions with increasing r. Also the allowed region is much larger for glycyl residues
than when a 8-carbon atom is present (about 6 times for normally allowed and
2.5 times for outer limit for T = 1100). Thus there is more freedom of rotation
about the single bonds when the amino acid residue at the a-carbon atom is glycine
than when it contains a p8-carbon atom.
Since the a-carbon atom is not an asymmetric carbon atom in the case of glycine,
no D or L isomer is possible. This is reflected clearly in Figs. 3a and b, which have
the symmetry of a center of inversion about (1800, 1800).
In order to verify how far the above results from theory agree with observation,
the parameters 4 and 4' were evaluated from the available crystal structure data on
di- and tripeptides, cyclic peptides, and polypeptides. These were calculated on
an IBM 1620 computer. In addition, the two values of 4)' (401' and 2'), which alone
are relevant in the case of amino acid structures, were also evaluated.
TABLE IV
AREA OF ALLOWED CONFORMATIONS EXPRESSED AS PERCENTAGE
OF THE TOTAL AREA OF THE ( -0') PLANE
T Normally allowed Outer limit
per cent per cent
(a) Non-glycyl residues
1050 3.5 12.7
1100 7.7 20.3
115' 8.8 24.2
(b) Glycyl residues
1100 45.1 57.4
1150 56.7 66.8

918 BIOPHYSICAL JOURNAL VOLUME 5 1965

 -s -"4 -3 2. . 3 4 5
n
FIGuRE 4a

Amino Acids. The two values 01', 42' for the various known crystal
structures of amino acids are given in Table V along with the values of the angle
N-aC-C (T). In general, 4' lies between + 30° and 1800 + 300, exceptions being
L-arginine HCl (Mazumdar, 1964), DL-aspartic acid (Dawson, 1953), L-glutamic
acid (Hirokawa, 1955), L-tyrosine HCl and L-tyrosine HBr (Srinivasan, 1959a
and b).
Di- and Tripeptides. In this case, for the N-terminal residues, c is the
only relevant parameter, since the positions of the hydrogen atoms attached to No
are not usually available. For the C-terminal residues, there is one value of t and
two values of 4/ corresponding to the two oxygen atoms of the C( group. For the
other residues (middle), there is one value of 4 and one value of 0'. These are
shown in Fig. 5, where the a-carbon atom under consideration is C1. The results are
given in Table VI along with the value of the angle N-aC-C' (T).
Considering the N-terminal residues, for which only k' is relevant, the observed

RAMAKIUSHNAN AND RAMACHANDRAN Polypeptide and Protein Chain Conformations 919.

180°
,* /#

values ofqb'liewl
\ :,5 s \ ~ ~0-SILK
~

andnon-bl
case ofglyyl
- -
<,

3ariwlwhhtnoeei
,,
A
/~~~~~~
"
.40

\ X
°
XL - LEFT4HANDED QXHELIX
dR- RIGHT-HANDED OC- HELIX
T -GAMMA HELIX
2 -27- HELIX
-R - RIBBON- STRUCTURE
~~~~~3-3 *010 HELIX
\ ~~~~~~4 _4 314 -HELIX
Jr -A[-HELIX
3W - C4J -HELIX

FIGUREPC

taloergoinTbI
reidues.In adition,# in
A/_POLY-GLY-PRO-HYPRO
~A -COLLAGEN

a
PUCKERED CAN

eneral lies etwee

vaueIo lie welwti4h.loedrgo etoe n al Iohih

casofgyy4n.o'lclreius nadto,O i eea,le ewe
180 *+30 geingwl ihtetedosre naioai tutrs
Bohfrgyy an n-glyy
the case ofC-terminal reides alotalte
and midcUe ofrainslewti
In residues, the conformations in~~~~~~~~~~~4
are plotted
Ftomhe4 4 aot

allowed
vleof4'lewlwihnthe
ntecsregion. fnngyy mentionediiner itablesIbthiinth
oe

920 BiopHysicAL JOURNAL VOLUME 5 1965

 FIGURE 4c
FIGURE 4 The normally allowed ( ) and the other limit (----) regions of
(n, h) for (a) T = 1050, (b) r = 110°, (c) T = 115°. The known polypeptide
and protein structures are also marked in Fig. 4b-® = left-handed a-helix; 6 =
right-handed a-helix; Q = gamma helix; © = 27-helix; ®g) = ribbon structure;
©0) = 3.010-helix; ) = 4.314-helix; i = r-helix; i = w-helix; A = poly-gly, -pro,
-hypro; A = collagen; 0 = silk;* = P-C puckered chain.

that six out of nine conformations listed in Table VI lie well within the normaly
allowed region and two more lie within the outer limit but outside the normally
allowed region. The one conformation that lies outside the allowed region is that
of the cysteine residue of the compound glutathione. But the value of r in this case
is 1150 and so the conformation comes into the allowed region corresponding to
7 = 1150 (Fig. 2c). In addition, the value of p' in many cases lies close to 1500,
which agrees well with the trend observed in amino acids.

RAMAKxISHNAN AND RAMACHANDRAN Polypeptide and Protein Chain Conformations 921

 TABLE V
OBSERVED VALUES OF 4o' IN AMINO ACIDS. THE VALUE OF THE
ANGLE N-aC-C' (T) IS ALSO GIVEN

Amino acid T ql' q2 Reference

degrees degrees degrees

DL-Alanine 108.4 162.9 -15.3 1
L-Arginine (in L-arginine dihydrate) 110.9 -169.3 12.1 2
*L-Arginine (in L-arginine HBr, H20) 109.5 -173.6 2.8 3
L-Arginine (in L-arginine HBr, H20) 110.2 -151.2 23.9 3
*L-Arginine (in L-arginine HCI, H20) 107.8 -174.2 1.7 3
L-Arginine (in L-arginine HCI, H20) 109.5 -154.3 22.6 3
*L-Arginine (in L-arginine HCI) 109.4 129.2 -45.7 3
L-Arginine (in L-arginine HCl) 108.0 139.6 -41.7 3
Asparagine (in asparagine monohydrate) 110.1 -168.8 6.1 4
DL-Aspartic acid (in DL-aspartic acid HCI) 109.7 -137.8 42.4 5
L-Cysteine (in L-cysteine HC) 105.2 169.1 -5.1 6
L-Cysteine (in s-methyl-L-cysteine sulphoxide) 111.1 176.7 -9.8 7
L-Cysteine (in hexagonal L-cysteine) 108.4 -165.8 12.3 8
L-Cystine (in L-cystine dihydrobromide) 108.1 -179.2 0.9 9
L-Glutamic acid 108.3 134.2 -31.9 10
DL-Glutamic acid (in DL-glutamic acid HCl) 110.7 -162.4 18.0 11
L-Glutamine (in glutathione) 110.6 170.5 -12.5 12
L-Glutamine 110.8 160.5 -11.4 13
Glycine (in bis glycino copper monohydrate) 111.3 171.7 -6.1 14
a-Glycine 111.8 -160.9 18.4 15
,6-Glycine 110.8 -152.7 23.8 16
7y-Glycine 110.8 -165.0 11.7 17
Histidine (in dihistidino zinc pentahydrate) 110.8 176.4 -3.9 18
Histidine (in dihistidino zinc dihydrate) 109.0 -168.2 9.1 19
Histidine (in histidine HCI, H20) 109.4 180.0 0.0 20
Hydroxy-L-proline II 110.7 -177.0 2.0 21
D-Isoleucine (in D-isoleucine HBr) 108.5 165.7 -20.6 22
L-Leucine (in L-leucine HBr) 110.1 160.4 -12.3 23
L-lysine (in L-lysine HCI) 109.7 160.1 -17.2 24
a-Methionine 112.1 146.3 -30.4 25
,j-Methionine 110.0 150.6 -31.6 25
DL-Norleucine 109.0 144.6 -24.4 26
Proline (in copper proline dihydrate) 109.7 -169.0 11.0 27
DL-Serine 110.1 -176.3 1.2 28
L-Threonine 110.4 153.9 -28.8 29
Tryptophan 111.9 175.8 21.4 30
L-Tyrosine (in L-tyrosine HCI) 110.1 142.1 -25.4 31
L-Tyrosine (in L-tyrosine HBr) 108.4 143.9 -30.4 32
L-Valine (in L-valine HBr) 108.1 168.4 -14.7 33
L-Valine (in L-valine HCI) 105.9 172.2 -8.9 34
L-Valine (in L-valine HCI, H20) 108.2 -173.1 5.3 35

*In the structures of L-arginine HBr, L-arginine HCl, H20, and L-arginine HC1, there are two mole-
cules per asymmetric unit.
References: (1) Donohue (1951); (2) Karle and Karle (1964); (3) Work done in Madras, Mazumdar
(1964); (4) Kartha and De Vries (1961); (5) Dr. B. Dawson, private communication; (6) Work done in

922 BIOPHYSICAL JOURNAL VOLUME 5 1965

 HiYI
I
vcg NI N\
N0 C1 C2

N-TERMINAL RESIDUE
Ho 0
CO% NoX C
i
_

-2
110 c.
00
C-TERMINAL RESIDUE

11 OIH
, CO NZ"'CI_.C'NI -,C
"'

I 2 FIGURE 5 The environment around the a-carbon

Ho 01 atom C1 in the case of N-terminal, C-terminal, and
MIDDLE RESIDUE middle residues of peptides.

Considering the glycyl residues, almost all of them lie within the allowed region.
Also in the case of C-terminal glycyl residues of the compounds Cys-Gly NaI and
Gly-Phe-Gly and in the case of middle and C-terminal residues in Gly-Gly-Gly
copper chloride, the observed conformations are such that they are allowed only
for glycyl residues.
Cyclic Peptides. Recently, the crystal structures of two cyclic hexapeptides,
cyclohexaglycyl hemihydrate (Karle and Karle, 1963), and Ferrichrome A (Zalkin,
Foster, and Templeton, 1964) have been reported. Of these, the former contains
four molecules per asymmetric unit and offers twenty four glycyl conformations for
investigation. The latter hexapeptide (Ferrichrome A) with the sequence Gly-Ser-Ser-
Orn-Orn-Orn (Orn standing for omithine) gives six conformations, one glycyl and
five non-glycyl. The parameters (S, 0') for these two hexapeptides are given in

Madras; (7) Hine (1962); (8) Oughton and Harrison (1959); (9) Peterson et al. (1960); (10) Hirokawa
(1955); (11) Dawson (1953); (12) Wright (1958); (13) Cochran and Penfold (1952); (14) Freeman
et al. (1964b); (15) Marsh (1958); (16) Itaka (1960); (17) Itaka (1961); (18) Harding and Cole (1963);
(19) Kretsinger and Cotton (1963); (20) Donohue and Caron (1964); (21) Donohue and Trueblood
(1952); (22) Trommel and Bijvoet (1954); (23) Subramanian (1965); (24) Wright and Marsh (1962);
(25) Mathieson (1952); (26) Mathieson (1953); (27) Mathieson and Welsh (1952); (28) Shoemaker
et al. (1953); (29) Shoemaker et al. (1951); (30) Work done in Madras; (31) Srinivasan (1959a);
(32) Srinivasan (1959b); (33) Parthasarathy (1962); (34) Work done in Madras; (35) Work done in
Madras.

RAMAKRISHNAN AND RAMACHANDRAN Polypeptide and Protein Chain Conformations 923

 TABLE VI
THE CONFORMATIONS (0, '9) OBSERVED IN DI- AND TRIPEPrIDES.
THE ANGLE N-aC-C' AT THE CORRESPONDING a-CARBON
ATOMS ARE ALSO LISTED
Peptides T X4 Reference
degrees degrees degrees degrees
N-acetyl glycine 110.4 1.5 -176.4 3.3 1
(a) Dipeptides
j3-Gly-Gly N-term-Gly 110.0 - -155.4 2
C-term-Gly 110.7 -0.8 -180.0 4.7 2
Gly-L-Asp N-term-Gly 111.2 - -171.4 3
C-term-Asp 109.5 69.0 -116.7 69.2 3
Gly-L-Tyr N-term-Gly 112.1 - 173.6 4
C-term-Tyr 111.4 76.6 146.6 -30.9 4
Gly-L-Tyr N-term-Gly 112.1 157.4 5
C-term-Tyr 112.0 106.3 146.7 -23.7 5
N-N' diglycyl N-term-Gly 109.0 -137.1 6
cystine C-term-Cys 109.5 16.5 178.8 -3.7 6
Cys-Gly sodium N-term-Cys 111.5 171.7 - 7
iodide C-term-Gly 125.7 -136.8 -147.6 33.1 7
Glutathione Middle-Cys 115.0 89.3 -2.8 - 8
('y-Glu-Cys-Gly) C-term-Gly 109.5 95.5 -169.1 10.0 8
p-toSyl-L-Pro-L- N-term-Pro 106.4 163.8 - 9
hypro C-term-Hypro 117.8 128.5 136.6 -43.6 9
L-Thr-L-Phe N-term-Thr 106.4 - 140.9 - 10
C-term-Phe 107.8 61.4 123.5 -67.0 10
L-Leu-Gly N-term-Leu 109.1 - -144.0 -11
C-term-Gly 112.3 86.4 176.6 2.9 11
(b) Tripeptides
N-term-Gly 107.9 169.4 12
Gly-Gly-Gly-CuC12 Middle-Gly 111.1 -65.6 133.4 - 12
C-term-Gly 110.7 -94.0 173.3 -4.7 12
N-term-Gly 109.9 - 126.8 13
Gly-L-Phe-Gly Middle-Phe 107.0 52.9 132.7 - 13
C-term-Gly 114.7 -96.5 175.4 -0.9 13
N-term-Leu 107.9 152.7 14
L-Leu-L-Pro-Gly Middle-Pro 111.3 112.2 162.5 - 14
C-term-Gly 114.5 3.7 178.1 0.5 14

*(1) Donohue and Marsh (1962); (2) Hughes and Moore (1949); (3) Pasternak et al. (1954); (4) Smith
and Wiebenga (1953); (5) Pasternak (1956); (6) Yakel and Hughes (1954); (7) Dyer (1951); (8) Wright
(1958); (9) Fridrichsons and Mathieson (1962); (10) Work done in Madras; (11) Work done in
Madras; (12) Freeman et al. (1964a); (13) Marsh and Glusker (1961); (14) Leung and Marsh (1958).

924 BIOPHYSICAL JOURNAL VOLUME 5 1965

Table V112 and are plotted in Fig. 2b. The glycyl conformations are also plotted
in Fig. 3a.
In the case of cyclohexaglycyl hemihydrate, a majority of the conformations lie
within the allowed regions. However, a few of them (1, 5, 7, 10, 12, 13, 15) are
clustered around 00 (or 3600) of i'. Although these conformations are not allowed
for 7 = 1100, they do become allowed for higher values of T (see Fig. 3b). It can be
TABLE VII
OBSERVED CONFORMATIONS (4,, 4,') IN CYCLOHEXAGLYCYL
HEMIHYDRATE AND IN FERRICHROME A
Residue 4 4,
degrees degrees degrees
Cyclohexaglycyl hemihydrate*
Gly (1) 112.8 85.6 9.9
Gly (2) 111.2 111.6 -29.2
Gly (3) 111.7 85.4 -88.9
Gly (4) 112.6 112.7 -29.3
Gly (5) 110.8 86.6 6.9
Gly (6) 113.0 115.6 -33.0
Gly(7) 112.1 86.8 5.8
Gly (8) 111.5 114.5 -32.6
Gly (9) 110.2 49.4 24.3
Gly (10) 116.2 89.7 -3.8
Gly (11) 110.0 48.2 26.3
Gly (12) 115.4 86.9 -1.6
Gly (13) 114.9 -103.1 16.9
Gly (14) 102.4 87.3 155.1
Gly (15) 113.7 -102.9 18.1
Gly (16) 105.3 -71.8 155.9
Gly (17) 105.3 -71.8 154.9
Gly (18) 109.5 -76.4 141.0
Gly (19) 109.7 -72.3 133.3
Gly (20) 105.3 -68.2 135.3
Gly (21) 110.2 65.0 -153.3
Gly (22) 108.3 60.1 -171.8
Gly (23) 110.0 95.7 -171.1
Gly (24) 109.5 104.1 -169.2
Ferrichrome At
Gly 114.7 -98.1 -1.2
Ser 1 114.2 123.7 132.3
Ser 2 104.0 17.3 171.5
Orn 1 113.5 71.6 6.6
Orn 2 113.2 101.4 -47.2
Orn 3 106.1 34.8 -160.7
Karle and Karle (1963); marked by the symbol, ], in Fig. 2b.
:Zalkin et al. (1963); marked by the open hexagon in Fig. 2b.
2 The coordinates of the atoms in the cyclic hexapeptide Ferrichrome A has kindly been fur-
nished by Dr. Zalkin to us prior to publication.

RAMAKRISHNAN AND RAMACHANDRAN Polypeptide and Protein Chain Conformations 925

seen from Table VII that for all these conformations, the value of T is systematically
greater than 1100 and thus these conformations also become allowed, under the
short contact criteria adopted in this paper.
In the case of the cyclic peptide Ferrichrome A, the conformations corresponding
to Ser 1, Ser 2, and Orn 1 lie well within the allowed region. Though the con-
formation corresponding to Orn 2 apparently lies outside the allowed region, it
is allowed on account of the higher value of T (1140) at the a-carbon atom. The
corresponding Orn 3 has a short contact Cp ... N2 according to the present study,
but in the actual structure this has a value of 2.96 A, which is greater than the
minimum value given in Table II. Thus this conformation is also allowed. The
conformation corresponding to the glycyl residue has a value of 1150 for T and
hence becomes allowed (Fig. 3b). An interesting feature of this conformation is
that it is allowed only for a glycyl residue. The value of q' for this conformation lies
close to 3600 which is also the case with many conformations in the previous cyclic
peptide.
Peptides in the Non-Helical Regions of Myoglobin. The structure of the
crystalline protein myoglobin contains long segments of a-helices linked by non-
helical peptide segments (Kendrew et al. 1960, 1961). The conformations at the
a-carbon atoms in these non-helical regions have been plotted by Dr. H. C. Watson
of Medical Research Council, Cambridge, England, who has kindly made the plot
available to the authors prior to publication. Most of the conformations lie within the
allowed regions for 7 = 1100, with a clustering of conformations around that of the
right-handed a-helix. A few are allowed only for 7 > 1100. In fact, two conformations
lie far outside the allowed limits, namely those of the residues EF3 and HC2. On
examination, it is found that the short contact responsible for disallowing those two
involve the pl-carbon atom, so that they will become allowed if they were glycyl
residues. When this fact was communicated to Dr. Watson, it was learned that these
were in fact giycyl residues, as expected. The others that lie just outside the allowed
regions have p' close to 00 (or 3600) and hence are likely to have a value of T
greater than 1100, in which case they also come within the allowed region.8
Polypeptides and Proteins. The conformations of the various structures
proposed for polypeptides and fibrous proteins have been calculated from the re-
ported coordinates of the atoms and are given in Table VIII along with the data on
the number of residues per turn, n, and the unit translation, h, along the axis of the
axis of the helix. These are plotted in Fig. 2b and also in Fig. 4b, which gives the
allowed combinations of (n, h) for = I1100.
All the structures except four (y-helix, 4.314-helix, 27-helix, and 2.27-helix) lie
within the allowed regions, some well within the normally allowed regions and some
others only within the outer limit region. These structures are discussed below.
8 The 0 - 0' diagram for myoglobin will be published shortly by Dr. Kendrew and Dr. Watson
(private communication).

-926 BIOPHYSICAL JOURNAL VOLUME 5 1965

 TABLE VIII
OBSERVED AND POSTULATED CONFORMATIONS (0, 4)') FOR POLYPEPTIDE
AND PROTEIN STRUCTURES. THE PARAMETERS
n AND h ARE ALSO LISTED
Polypeptide or protein T ) o' n h Reference*
degrees degrees degrees A
a-helix (3.61s) 109.5 133.0 -57.2 3.615 1.495 1
'y-helix (5.117) 110.1 -96.3 78.0 5.143 0.98 2
27a-helix 111.3 105.1 69.5 2.000 2.80 1
2.27-helix (ribbon structure) 111.6 101.9 59.2 2.169 2.75 3
3.010rhelix 111.6 130.7 -25.7 3.000 2.00 3
4.314-helix 100.5 -91.9 91.7 4.337 1.20 3
'-helix (4.418) 114.9 122.9 -69.7 4.40 1.15 4
Polyglycine II 109.1 102.0 145.8 -3.00 3.10 5
Poly-L-proline II 110.0 102.8 145.9 -3.00 3.12 6
Poly-L-proline II 108.8 103.7 145.1 -3.00 3.12 7
Poly-L-hydroxyproline A 105.5 103.1 147.6 -3.00 3.05 8
co-form of poly-jS-benzyl-
L-aspartate 109.9 -115.6 55.4 -4.00 1.325 9
Collagen type helix 110 116 145 -3.28 2.95 10
Silk 110 40 135 2.00 3.45 11
P-C puckered chain 110 57 118 2.00 3.3 12

*(1) Bamford et al. (1956); (2) Pauling and Corey (1951); (3) Donohue (1953); (4) Low and Grenville-
Wells (1953); (5) Crick and Rich (1955); (6) Sasisekharan (1959a); (7) Cowan and McGavin (1955);
(8) Sasisekharan (1959b); (9) Bradbury et al. (1962); (10) Ramachandran (1963); (11) Marsh et al.
(1955); (12) Pauling and Corey (1953).

a-helix. Both the right-handed a-helix (1320, -57°) and the left-handed
a-helix (-132°, 570) lie within the outer limit regions only. However, slight
perturbations such as changing the value of r would bring the right-handed helix
into the normally allowed region but the left-hand a-helix is always outside the
normal limits although it is just allowed by the outer limits. Also the right-handed
a-helix can accommodate L-proline towards an end, since f' 1200, while the
left-handed a-helix cannot. Even in the former case, because the planes of the
peptide groups are nearly vertical, short contacts arise between the imino carbon
atom, C, and the atoms of the backbone of the residue on top of it, if it is in the
middle. It has been found that the two a-helical portions adjoining the proline ring
must be inclined by at least 350 in order that these short contacts may be relieved.
Even in this case, there arise two short contacts C2' . . . C1' = 2.61 A and
C2 ... 01 = 2.45 A between atoms in the adjacent residues and these can be
relieved only by distorting the planarity of the peptide group.
y-helix. The conformation of the right-handed y-helix (-96°, 780) lies
well outside the allowed region and hence is not likely to be observed.

RAMAxRSHNAN AND RAMACHANDRAN Polypeptide and Protein Chain Conformations 927

 27a-helix. This type of folding originally proposed by Huggins (1943)
and designated as 27-helix by Bragg et al. (1950) has the conformation (1050, 700)
and lies apparently outside the allowed region. But the short contact in this case (as
well as in the case of the 2.27-helix to be discussed next) is 01 *H2 (1.93A). Since
..

this distance forms part of a possible intrachain hydrogen bond with N2 .01 =
2.72 A and H2N201 = 28'30', this is really not a short contact.
2.27-helix (ribbon structure). This structure proposed by Donohue (1953)
has the conformation (1020, 590) and this also apparently lies outside the allowed
region. As in the previous case, the short contact 01 H2 (1.88 A) forms part
..

of a possible intrachain hydrogen bond and so this conformation becomes allowed.

In fact a similar type of folding has been observed by Kakudo et al. (1963) in a
tetrapeptide.
3.010-helix. This structure proposed by Bragg et al. (1950) and Donohue
(1953) has the conformation (1310, -260) for the right-handed helix and lies
close to the a-helix conformation, within the outer limit region. This structure is
also quite likely to occur.
4.314-helix. This structure, also proposed by Donohue (1953), has the
conformation (-92°, 920) for the right-handed helix and lies close to the y-helix,
outside the allowed regions and hence is not likely to occur.
4.416-helix (7r). The two slightly different forms of 7-helices proposed by
Low and Grenville-Wells (1953), one with n = 4.3 and h = 1.14 A and the other
with n = 4.4 and h = 1.15 A, have the conformations (1240, -69°) and (1230,
-700) lying close to the conformation of a-helix and are quite likely to occur.
Poly-Gly, -Pro, -Hypro. The conformations of these helices (left-handed)
lie close to (1000, 1500) well within the normally allowed region. In the case of
poly-L-proline and poly-L-hydroxyproline, the value of 4 is necessarily close to
1200 for the imino residues to be accommodated. Also, the right-handed counter-
parts of these cannot accommodate imino acid residues, since the value of 4, is close
to -120° in these cases.
w-Form of Poly-f3-Benzyl-L-Aspartate. The structure of the 0-form of
poly-B-benzyl-L-aspartate has been determined by Bradbury et al. (1962) to be a
left-handed helix with a fourfold screw axis and with a folding similar to the 418.-
helix proposed by Bragg et al. (1950). The conformation of this helix is (-116°,
550) and this lies just outside the allowed region. However, by making the peptide
groups non-planar, the short contact that disallows this conformation, namely
C .
C1', can be made to have a value of 3.13 A, which is allowed by the outer
limit. Thus this structure, in the form proposed, also becomes allowed.
Silk. Silk has the conformation close to (400, 1350) which lies well within
the allowed region. The structure is slightly buckled with h = 3.45 A, smaller than
that of a fully extended chain (h = 3.63 A). This is due to the presence of side
chains like alanine, etc. In fact, the conformations of other fl-forms of polypeptides

928 BIoPHYsIcAL JoURNAL VOLUME 5 1965

like polyglycine, /3-poly-L-alanine, fl-poly-y-methyl-L-glutamate have conformations
close to silk and are also slightly bucked.
P-C Puckered Chain. This chain conformation proposed by Pauling and
Corey (1953) for the pl-form of proteins has the conformation (570, 1180), close
to that of silk. But this is much more buckled and the value of h in these cases is
3.3 A, less than that for silk (3.45 A). This is fully allowed, but this chain cannot
accommodate proline and this has led to the suggestion of an alternative puckered
chain (Pauling and Corey, 1953; Ramachandran, 1962; Sasisekharan, 1962).
Collagen. The polypeptide chains of the collagen helix (n =-3.28 and
h = 2.95 A) has the conformation (1160, 1450). Because 4 1200, imino acid
residues can be readily accommodated. The value of 4' close to 1800 is also a
natural occurrence in the case of amino acids. Consequently, the fact that this
conformation provides enough sites for imino acids and that it has a natural value of
4)' suggest that, even without internal hydrogen bonding, the collagen chain con-
formation is likely to be quite stable.
The conformation of collagen lies within the normally allowed region. Another
interesting feature is that if 4' is kept close to 1200, the value of h does not vary
from 2.9 A by more than 5 per cent within the allowed region, thus explaining the
relative inextensibility of collagen.

CONCLUDING REMARKS
Thus the conformations observed in simple and cyclic peptides are seen to confirm
the predictions of the theory, thereby proving the validity of the stereochemical
criteria assumed in this study.
Finally, the results obtained in our study agree well with those obtained by
others from considerations of potential energy. Thus, Mizushima and Shimanouchi
(1961), from a study of the internal rotation of simple molecules have preferred
nine orientations. In our notation, these correspond to 4) = 0, 120, and 2400,
)' = 60, 180, and 3000. These are given in Table IX, along with the values of the
unit twist t and the unit translation h. An examination of these in the light of the
present study shows that all except d, e, and i lie within the allowed region. Even
among these conformations, i becomes allowed because of the formation of a hydro-
gen bond of the type N2-H2 ... 01, which offsets the short contact, thus leaving the
conformation d and e alone to be really disallowed.
Recently, De Santis et al. (1963) have attempted a study of the stability of
helical conformations of linear polypeptide chains from potential energy considera-
tions. Although there is a broad similarity between their potential energy contours
and the boundaries of our diagrams, there are some differences, for example a deep
potential mini'mum corresponding to about (1200, 300°) and another one at about
(1600, 600). Neither of these are particularly populated in Fig. 2b, showing that

RAMAKRISHNAN AND RAMACHANDRAN Polypeptide and Protein Chain Conformations 929

 TABLE IX
PREFERRED ORIENTATIONS OF POLYPEPTIDE CHAIN IN OUR
NOTATION (ACCORDING TO MIZUSHIMA AND SHIMANOUCHI)
Twist angles No. of residues Residue
Unit twist per turn height
Conformation + t n h
degrees degrees A
a 0 180 180 2.00 3.63
b 0 -60 -96 -3.75 2.45
c 0 60 96 3.75 2.45
d 240 180 84 4.28 2.72
e 240 -60 176 2.05 2.63
f 240 60 -90 -4.00 1.16
g 120 180 -84 -4.28 2.72
h 120 -60 90 4.00 1.16
i 120 60 -176 -2.05 2.63

for a single pair of residues these are not specially favored. The potential energy
data that are fed in such studies may have to be further revised.

APPENDIX
While evaluating the contact distances for non-glycyl residues, only the pl-carbon atom
of the side chain was taken into account. It has been possible to extend the study to
working out the effect of the y-carbon atom on the conformation of the two residues.
This was made possible since it has been found in Madras, from an analysis of the
observed orientation of the side chains in the structures of various amino acids and
peptides, that there are three preferred positions for the y-carbon atom.
The position of C, may be represented by a parameter + which measures the rotation
of C, about the C6-C, bond from a standard initial conformation (& = 00) in which
the atom C, lies in the plane N-Ca-Cp and with atoms N and C, being in the cis configura-
tion with respect to the C.-Cp bond. Viewing from Cp to C., & is taken to be positive for
an counterclockwise rotation. With this convention, the three positions of C, are found
to lie close to values of 60, 180, and 3000 for b, and these are indicated by indices 1, 2,
and 3 respectively.
The contact distances between the y-carbon atom in these three positions and the back-
bone atoms were calculated in the range of (4), 4') allowed by the ,8-carbon atom (given
in Table III) for r = 1100. The alterations in the outer limit boundaries thus worked
out are shown in Fig. 6, where the shifts in the boundary due to the y-carbon atom in the
three positions are shown by arrows.
In all the three cases, there are some additional restrictions in the allowed ranges of
either 4 or 4', or both, as can be seen from Fig. 6. For the first position of C, (v = 600),
there are restrictions on both 4 and 4', while for the second position (& = 1800), the
restrictions are on 4' only and for the third position (& = 3000), on 4 only.
Fig. 6 also contains the conformations of the right-handed and left-handed a-helices
marked in it. It is interesting to note that for the first position C, (g = 600) the con-

930 BIOPHYSICAL JOURNAL VOLUME 5 1965

 360

3BC

OP ISO*18 36d
FIouRB 6 The shifts in the boundaries (indicated by .) of the allowed regions
(outer limit) taking into account the restrictions due to 'y-carbon atom in the three
positions: C, alone; ---- with C, in position 1; .... .... C, in position 2;
and -.-.-.-. C, in position 3. The conformations of the right- and left-handed a-
helices are also marked.

formation of the right-handed a-helix lies just on the boundary of the outer limit region,
while the conformation of the left-handed a-helix goes outside the outer limit. In the
other two cases, both are only within the outer limits and there is no obvious case for
either the right-handed or the left-handed a-helix to be more likely. In the analysis made
on amino acid side group conformations (Lakshminarayanan, unpublished), both
L-glutamic acid and L-glutamic acid HCI have the y-carbon atom in position 1, which
may explain why poly-L-glutamic acid has a hight-handed helix. However, the three
positions are found to occur fairly evenly distributed in different amino acids, and this
explanation will not hold good for the very common occurrence of the right-handed alpha
helix in preference to the left-handed one in various polypeptides.
Our thanks are due to Dr. V. Sasisekharan for the discussions which we had with him. One of
the authors (C.R.) wishes to thank the Ministry of Scientific Research and Cultural Affairs
and The Council of Scientific and Industrial Research, India, for financial support. The other
author (G.N.R.) is grateful to the United States National Science Foundation for a Senior
Visiting Fellowship, which enabled him to spend a year in the University of Michigan. We are
very grateful to Professor J. L. Oncley for his interest in these studies.
Part I of this article appeared in Proc. Indian Acad. Sc., 1964, A59, 327.
Received for publication, July 19, 1965.

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