BIOCHIMICA ET BIOPHYSICA ACTA

BBA 35666

STUDIES ON T H E CONFORMATION OF AMINO ACIDS"

VII. BACKBONE AND SIDE-CHAIN CONFORMATIONS OF N-TERMINAL

RESIDUES IN PEPTIDES

I53

P. K. P O N N U S W A M Y AND V. S A S I S E K H A R A N

Centre of Advanced Study in Biophysics, University of Madras, Madras-25 (India)

(Received March 2nd, 197 o) (Revised m a n u s c r i p t received J u l y I3th, 197 o)

SUMMARY

Potential energy calculations have been made to predict the preferred confor- mations of N-terminal residues in polypeptide chains. Nonbonded, electrostatic and torsional energies have been computed using appropriate energy functions and their role assessed. The effects of fl-, 7-, and 8-atoms in the side chain on the backbone conformation are discussed. A conformation in which the plane of the peptide group is coplanar with the plane through the atoms N, C a and C' is preferred for the N- telminal glycyl residue, and this coplanarity is affected by the presence of/5- and v-atoms only and not beyond. The side-chain conformations about the Ca-C~ and C~-Cv bonds are nearly the same as those predicted for the corresponding free amino acid examples. The observed conformations of N-terminal residues in the crystal structures are compared with the theoretical predictions and the agreement is found to be good.

INTRODUCTION

In the earlier parts of this series 1-4 we dealt with the energies of the confor- mations of amino acids with different side chains and discussed the probable confor- mations of their backbones and the side chains. For this purpose, conformational energies were obtained classically using the Lennard-Jones type of potential function a. In this and in the subsequent paper, we deal with the energy calculations for the N- terminal and C-terminal residues, respectively, in peptides with representative side groups so that the observed conformations of peptides and proteins could be explained. A preliminary account of the backbone conformations of the N- and C-terminal glycyl and alanyl residues has already been reported s.

As in the earlier papers of this series 1-4, the potential energy of conformations has been expressed in terms of the angles of the torsion about the various single bonds.

" Con t r ibu t ion No. 297 f rom t he Centre of A d v a n c e d S t u d y in Bio-Physics , Un ive r s i t y of Madras , Madras-25, India .

Biochim. Biophys. Acta, 221 (197o) i 5 3 - i 5 8

T54 P . K . PONNUSWAMY, V. SASISEKHARAN

The non bonded, the torsional and the electrostatic contributions were individually obtained and the sum was taken to be the total energy. The nonbonded energy was computed using the ' 6 12' function taking into account all pairs of nonbonded atoms whose distances va ry with the rotation. A threefold barrier of height 2.8 kcal/mole was used throughout about the bonds of the type C(sp a) C(sp 3) and no barrier for the rotat ions about the C(sp 3) C(sp 2) bonds. The residual charges on the various atoms in each molecule were obtained from quan tum mechanical considerations as described by RENUGOPALAKRISHNAN t?t al. 6. The dielectric constant a" was varied from 2 to io to see the effect of the variation on the energy distribution. I t was found that irre- spective of e, the theoretically predicted minimum remained nearly the same ; however, the energy surface near the min imum occupied different regions.

The calculations were made on rigid models built with the average dimensions for the N- and C-terminal residues taken from A. V. Lakshminarayanan and V. Sasise- kharan (private communicat ion) ; the conformation itself being characterized by the relevant rotat ional parameters such as q~, ~, Z' , Z 2, etc.

NOTATION AND DEFINITIONS

In defining the conformational parameters, the conventions proposed earlier by EDSALL et al. 7-9 and described in detail by RAMACHANDRAN AND SASISEKHARAN 5 have been followed. These parameters are shown in Fig. I for a typical case. The conformational angles are taken to be positive for a clockwise rotat ion and qv goes from o to I2O ° only. The neutral form of the amino group has not been considered, as the amino group is alway.~; found to be T + NH 3 in all the observed crystal s tructures of the peptides ~°.

RESULTS AND DISCUSSION

The conformational energies have been calculated for the glycyl, alanyl, butyl , leucyl and aromatic side-group residues as a function of the relevant rotat ional parameters 9~, ~0 and Z. The methyl hydrogens at the y-(butyl) and at the &(norvalyl

d

O~~a ~ ~4~\ i H~I

14 ~X~ !4 L4"f~'

\ \

g

Fig. L The confo rma t iona l p a r a m e t e r s (F, ~/~ and Z 1 for the N- t e rmina l bu ty l residue.

Biochim. Biophys. dcta, 22~ (i97o) I 5 3 - I 5 8

CONFORMATIONS OF ~ - T E R M I N A L RESIDUES 155

T A B L E I

POSITIONS OF MINIMA AND THE PROBABLE RANGES IN /fl (SHOWN IN PARENTHESES) (AND IN ZZ FOR AROMATIC SIDE; GROUP)

Residue qo ~p Z 1 Z* Energy Remarks (Kcal/mole)

Non- Total bonded

Glycyl 60 IiO (6o-13 o) o.oo Not f avoured 60 25 ° (23o-300) o.oo No t f avoured 6o o (32o-4o) o.oo F a v o u r e d

Alanyl 7 ° 12o (i 15-15o ) o.oo No t f avoured 60 34 ° (24o-5) 0.08 F a v o u r e d 60 34 ° (32o-5) o.oo F a v o u r e d

B u t y l 8o 15o 6o 1.94 9.5 ° Not favoured 5 ° 34 ° (33o--5) 60 0.27 o.oo F a v o u r e d 7 ° 12o 18o o . io 6.41 Not f avoured 60 320 (315-335) 19o o.oo 0.58 F a v o u r e d 7 ° 12o 290 0.08 5-95 Not f avoured 7 ° 34 ° (33o-5) 29o o.31 0.22 F a v o u r e d

Norva ly l 7 ° 12o 18o 7 ° 0.28 3.00 Not favoured 7 ° 320 (305-335) 18o 7 ° o.oo o.oo F a v o u r e d 7 ° 14o 60 18o 3.63 6.96 Not f avoured 5 ° 34 ° (325-5) 5 ° 17o 0.55 o.o6 F a v o u r e d 60 12o 19o 17o 0.65 3.41 Not favoured 60 320 (315-345) 19o 17o 0.26 o . n F a v o u r e d 7 ° 12o 30o 19o 0.5o 3.20 Not f avoured 7 ° 34 ° (32o-5) 30o 19o 0.63 0.23 F a v o u r e d 7 ° 12o 31o 3oo 0.5o 3.1o Not f avoured 7 ° 34 ° (31o-5) 31o 300 0.49 o.16 F a v o u r e d

Leucyl 7 ° 12o 17o 60 o.oo 2.72 Not f avoured 60 31o (305-34 ° ) 17o 6o o.18 0.07 F a v o u r e d 7 ° 12o 31o 18o 0.48 2.88 Not favoured 7 ° 34 ° (29O-lO) 31o 18o 0.50 o.oo F a v o u r e d

Aroma t i c 60 34 ° (32o-5) 60 90 (75-1o5) o.oo o.I i F a v o u r e d 60 12o 18o 8o 0.24 3.07 N o t f avoured 6o 34 ° (30o-345) 18o 8o (5O-lO5) o.17 o.oo F a v o u r e d 60 12o 3o0 IOO o.80 3.44 Not favoured 60 34 ° (325-36o) 3oo IOO (75-135) 0.78 0.34 F a v o u r e d

a n d l e u c y l ) c a r b o n s w e r e k e p t f i x e d a t t h e s t a g g e r e d c o n f o r m a t i o n s . T h e a r o m a t i c

s i d e g r o u p s o f p h e n y l a l a n y l , t r y p t o p h a n y l a n d h i s t i d i n y l r e s i d u e s w e r e r e p r e s e n t e d

b y t h e p l a n a r g r o u p - C v (C6H, C e l l ) o r t h e o n e - C v ( N e l l , C6H) a s w a s d o n e i n t h e

f r e e a m i n o a c i d e x a m p l e s 4.

I n T a b l e I t h e l o w e n e r g y c o n f o r m a t i o n s a n d t h e i r r e l a t i v e e n e r g y v a l u e s i n

e a c h o f t h e r e s i d u e s a r e g i v e n , a l o n g w i t h t h e c o r r e s p o n d i n g c o n f o r m a t i o n a l p a r a m e t e r s .

T h e p r o b a b l e r a n g e s * i n t h e p a r a m e t e r s ~o a n d Z ~ ( in t h e a r o m a t i c c a s e a l o n e ) f o r e = 4

a r e a l s o g i v e n i n t h i s t a b l e .

* The range covered b y t he 0. 5 kcal /mole energy con tour wi th respec t to the m i n i m u m is referred to as t he probable range of a ro ta t iona l pa rame te r . See also P a r t II . Th i s is followed in t he s u b s e q u e n t pape r s also.

Biochim. Biophys. Acta, 22i (197 o) i 5 3 - I 5 8

156 P. K. PONNUSWAMY, V. SASISEKHARAN

(a) (b)

N N,O

N 0

N

N 0 N

N (c) (d)

0 N

• I l "\ t

0 ( 5 : ~, ',

(e)

Fig. 2. Projection down the C"-C' bond corresponding to (a) nonbonded and (b) total minimal energy conformat ions for the N-terminal glycyl residue. (c) and (d), respectively, are the corre- sponding conformat ions for the alanyl residue. (e) projection of N-terminal buty l residue to show" close contact between Ct~ and NH, and between Cv and NH groups when Z ~ - 18o ~' and ~p o '

Backbone conformation The nonbonded energy a lways favours a conformat ion near (6o °, i I o I2(¢)

(The first number denotes ~v and the second ~v). This conformat ion is shown in projec t ion (Newman) in Figs. 2a and c. The t e rmina l amino hydrogens are s taggered abou t the N - C a bond, and the C 0 bond is a p p r o x i m a t e l y eclipsed by the Ca-H a bond. However , this conformat ion is not observed in the solid s ta te , and hence is marked as "no t f avoured" in Table I. The inclusion of the e lec t ros ta t ic energy shifts the min imal energy conformat ion to (6o °, o °) for tile g lycyl and to (60 °, 34 o°) for the a lanyl and for the o ther nonglycyl cases as shown in Figs. 2b and d. These correspond closely to the conformat ions ac tua l ly observed in the g lycyl and the nonglycyl residues in the solid s ta te , and hence are m a r k e d as " favoured" in Table I. I t will be not iced t ha t the differ- ence be tween the equi l ibr ium conformat ions of the g lycyl and the nonglycyl residues is tha t , while in the g lycy l case the m i n i m u m occurs at ~0 = o ° (the C~ O bond eclipses the N C a bond), in the a lanyl and o ther nonglycyl cases the m i n i m u m occurs at a lower ~0 value, namely below 34 °0 ( C = O and N-C a bonds are not eclipsed). The s t ab i l i t y of the backbone conformat ion (6o °, 34o-32o °) is ma in ly due to the e lec t ros ta t ic a t t r a c t i on be tween the amino group and the ca rbonyl oxygen. The occurrence of the m i n i m u m at ~o - - 34 °o for the a lanyl and o ther nonglycyl residues (except for the Posi t ion I I of the ),-atom), ins tead of a t o °, is ma in ly due to the s ter ic repuls ion be- tween the ni t rogen and the Ca-atoms at ~ = o °. In Posi t ion I I of the ) ,-atom, there is fur ther s ter ic h indrance be tween the ) , -a tom and the imino group of the pep t ide uni t a t ~o = 34 ° 36o ° (see Fig. 2e) and therefore the equi l ibr ium occurs near to ~0 ~:-- 32o °. Tab le I shows t ha t the backbone conformat ion is not affected b y the a toms beyond the ) , -atom in the side chains, i.e., the prefer red backbone conformat ions are near ly the same as t ha t of the b u t y l residue.

Table I I gives the observed % ~0, Z ~ and Z ~ values in the r epor ted crys ta l struc- tures of N- t e rmina l residues. The observed ~ and ~0 values of the g lycyl and the non-

Biochim. Biophys. Acta, 221 (i97o) ~53-I58

CONFORMATIONS OF N-TERMINAL RESIDUES

TABLE II

THE OBSERVED ~7, ~0, ~ l AND ~2 VALUES OF N-TERMINAL RESIDUES*

I57

Structure ~o ~ Z1 •2

Glycyl fl-Gly-Gly Gly-Gly HC1 H20 Gly-Asn Gly-Tyr HC1 H20 N,N'-Digly-Cys SS Cys 2H~O Gly-Phe-Gly H20 P-bromocarbobenzoxy Gly-Pro-Leu-Gly

Nonglycyl Leu-Gly HBr Thr-Phe-Nitrobenzyl ester HBr P-Tosyl-Pro-Hyp Leu-Pro-Gly H20

Lysozyme** Lys

Chymotrypsin** Cys

336.2 51.1 341.4

351.3 349.8

76.1 12.8 73.7 3o7 .6

350.5

329.8 292.8 155.2 321.7 175.2 337.9 333.4 279.4 17o.3

334.0 175.o 195.o

337.0 75.o

* The values are taken from LAKSHMINARAYANAN et al. n. References to the structures are not given here.

** The values were taken from refs. 12, 13 and 14, respectively.

glycyl examples agree well with the corresponding values predicted by the theory. In particular, it may be noted that in the crystal structures of non-glycyl residues, ~v lies between 320 ° and 34 o°. This is in very good agreement with the theory which demands deviations of this magnitude from the near coplanar arrangement of the peptide plane and the plane passing through atoms N, C a and C'.

Side-chain conformations Regard ing the s ide-chain conformat ions , i t will be not iced from Table I t h a t

i r respect ive of the na tu re of the a toms beyond the 9'-atom there appea r three local min ima a round Z 1 nea r ly 60 °, 18o ° and 300 ° corresponding to the three s taggered posi t ions of the 9'-atom abou t the Ca-C~ bond. The three posi t ions appear to be a lmost equal ly preferable. Fo r the norva ly l residue X 2 is res t r ic ted to near ly 60 °, 18o ° and 300°; however not all the nine (X 1, Z*) s taggered combina t ions are favourable . As in the free amino acid case 4 the following five combinat ions , namely , (9'1, ~II) , (9'11, ~I), (9'1I, ~II) , (9'11I, 8II) and (9,111, ~III)* were alone in the low energy regions and the r ema inde r in the re la t ive ly higher energy regions, owing to s ter ic repuls ion be tween e i ther the d -methy l and the pep t ide n i t rogen or the 8 -methy l and the ca rbonyl carbon as the case m a y be. All five preferred combina t ions appea r to be equa l ly favourable . However , for the leucyl side chain, combina t ions (y l I , 81) and (9'111, 811) are more favoured t han the o ther combinat ions . These two prefer red combina t ions are the same as ob ta ined for the free leucine molecule 4. In the a romat ic side group, in general,

* In this notation, for example, yI means that the y-atom is in Position I with Z 1 around 60 °, and y l I means that the y-atom is in Position II with Z ~ around 18o °.

Biochim. Biophys. Acta, 221 (197 o) 153-158

I 5 ~ P. K. PONNUSWAMY, V. SASISEKHARAN

an orientation of the ring system nearly perpendicular to the plane through the atoms Ca, C# and Cv was preferred for each of the three positions of the v-atom. The characteristic probable ranges in ;g2 were nearly the same as those predicted for the free amino acid example 4.

Conclusion The following are the main conclusions drawn from the excellent agreement

between theory and observation. (I) The backbone conformation of N-terminal residues with different side chains

varies little. The equilibrium backbone conformation is the one in which the plane of the amide group and the plane through the atoms N, C a, C' are nearly coplanar; the presence of C# necessitates a deviation of 20 °, and C v in Position I I a deviation of about 4 o°, from the coplanar arrangement.

(2) The side-chain conformations beyond the v-atom appear to he independent of the preferred backbone conformation, and are nearly the same as for the free amino acids.

ACKNOWLEDGEMENTS

We thank Professor G. N. Ramachandran for his interest. One of us (P.K,P.) thanks the University Grants Commission (India) for financial assistance. This work was partially supported by U.S. Public Health Service Grant No. AM Io9o 5.

R E F E R E N C E S

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the press. 5 O. N. RAMACHANDRAN AND V. SASISEKHARAN, Advan. Protein Chem., 23 (1968) 283. 6 V. RENUGOPALAKRISHNAN, A. V. LAKSHMINARAYANAN AND V. SASISEKHARAN, in preparation. 7 J- T. EDSALL, P. J. FLORY, J. C. KENDRE\V, A. M. LtQUORI, G. NEMETHY, G. N. RAMA-

CHANDRAN AND H. A. SCHERAGA, J . Mol. Biol., 15 (1966) 399. 8 J. T. EDSALL, P. J. FLORY, J. c. KENDREW, A. M. LIQUORI, G. NEMETHY, G. N. RAMA-

CttANDRAN AND H. A. SCHERAGA, J . Biol. Chem., 241 (1966) lOO 4. 9 J- T. EDSALL, P. J. FLORY, J. c. I~ENDREW, A. M. LIQUORI, G. NEMETHY, (;. N. RAMA-

CHANDRAN AND H. A. SQHERAGA, Biopolymers, 4 (1966) 12r. IO R. E, MARSH AND J. DONOHUE, Advan. Protein Chem., 22 (1967) 234. I I A. V. LAKSHMINARAYANAN, V. SASISEKHARAN AND G. 12",1. RAMACHANDRAN, in (;. N. RAMA-

CHANDRAN, Conformation of Biopolymers, Vol. I, Academic Press, London, 1967, p. 61. 12 C. C. F. BLAKE, D. F. KOENIG, G. A. MAIR, A. C. T. NORTH, D. C. PHILLIPS AND V. I(. SARMA,

Nature, 206 (1965) 757. 13 C. C. F. BLAKE, D. F. ]A:OENIG, G. A. MAIR, A. C. T. NORTH, I). (3. PHILLIPS AND V. It. SARMA,

Proc. Roy. Soc. London, Ser. 13, 167 (1967) 365. 14 J. J. BIRKTOFT, B. V~ r. ,~/[ATTHEWS AND D. M. BLO~V, Biochem. 13iophys. Res. Commun., 36

(1969) 131.

Biochim. Biophys. Acta, 221 (197 o) 153-158