Theoretical conformational analysis of the bovine adrenal medulla

12 residue peptide molecule

N.A. Akhmedova,*, Z.H. Tagiyevb, E.M. Hasanovb, G.A. Akverdievaa

aMolecular Biophysics Laboratory, Baku State University, Z. Khalilov str. 23, Baku 370148, AzerbaijanbAzerbaijan Medical University, Bakichanov str. 23, Baku 370022, Azerbaijan

Received 5 June 2002; revised 23 October 2002; accepted 8 November 2002

Abstract

The spatial structure and conformational properties of the bovine adrenal medulla 12 residue peptide Tyr1-Gly2-Gly3-Phe4-

Met5-Arg6-Arg7-Val8-Gly9-Arg10-Pro11-Glu12 (BAM-12P) molecule were studied by theoretical conformational analysis. It

is revealed that this molecule can exist in several stable states. The energy and geometrical parameters for the low-energy

conformations are obtained. The conformationally rigid and labile segments of this molecule were revealed.

q 2002 Elsevier Science B.V. All rights reserved.

Keywords: Conformational analysis; Structure; Function; Peptide

1. Introduction

The opioid dodecapeptid Tyr1-Gly2-Gly3-Phe4-

Met5-Arg6-Arg7-Val8-Gly9-Arg10-Pro11-Glu12

signed as BAM-12P (bovine adrenal medulla 12

residue peptide) was extracted from the medulla of

bovine adrenal. The opiate activity of BAM-12P was

2,4 times higher than the activity of Met-enkephalin

and 1,8 times higher than the activity of b-endorphin

[1]. The sequences of residues 1–5 and 1–8 of BAM-

12P correspond to Met-enkephalin and adrenorphin,

respectively, which are highly potent as an opiate [2,3].

BAM-12P, in turn, is a fragment of the residues 1–12

and 15–26 of the opioid peptides E and I, respectively

[4–6]. All mentioned peptides are the products of the

degradation of the bovine precursor pro-enkephalin

[7]. BAM-12P plays important role in the functioning

of the central nervous system, because of that it is

considered as neuropeptide. Apart from analgetic

effect, such peptides show other properties, as a

substantiating, psychotropic action, a participation in

a regulation of a visceral system of an organism,

namely, exchange processes, a systems of digestion, a

pancreas and cardiovascular system [8].

The diversity of biological functions of given

peptide is undoubtedly connected to its confor-

mational possibilities. In order to elucidate the

mechanism of action of the peptide the investigation

of the structure-function relationship is necessary,

that first of all requires the information about of the

full set of low energy and consequently the

potentially and physiologically active conformations

of this molecule.

0022-2860/03/$ - see front matter q 2002 Elsevier Science B.V. All rights reserved.

PII: S0 02 2 -2 86 0 (0 2) 00 5 79 -3

Journal of Molecular Structure 646 (2003) 75–80

www.elsevier.com/locate/molstruc

* Corresponding author. Tel.: þ994-12-39-03-05.

E-mail address: hagverdieva@hotmail.com (N.A. Akhmedov).

The present paper discuss the spatial structure of

BAM-12P molecule.

2. Method

The investigations were carried out on the basis of

theory and method the theoretical conformational

analysis elaborated and described in Refs. [9–12].

The conformational potential energy of system is

selected as sum of the independent contributions of

nonvalent (Env), electrostatic (Eel), torsional (Etors)

interactions and the energy of hydrogen bonds (Ehb).

The nonvalent interactions were calculated by the

Lennard–Jones potential with the parameters pro-

posed by Scott and Sheraga [13]. The contribution of

electrostatic interactions was taken into account in a

monopole approximation with partial charges of atoms

as suggested by Scott and Sheraga [13]. The effective

dielectric constant 1; length of valent bonds and the

valent angles, the potentials and barriers for the

torsional interaction calculation were taken from

the works [14,15]. The values of used internal barriers

are represented below: for the backbone chain Uw0 ¼

2:5 kJ=mol; Uc0 ¼ 0:8 kJ=mol; Uv

0 ¼ 83:7 kJ=mol; for

the side chains, Tyr: Ux10 ¼ 12:6 kJ=mol; U

x20 ¼ 0:8 �

kJ=mol; Ux30 ¼ 83:7 kJ=mol; Phe: U

x10 ¼ 12:6 kJ=mol;

Ux20 ¼ 0:8 kJ=mol; U

x30 ¼ 8:4 kJ=mol; Met: U

x10 ¼

12:6 kJ=mol; Ux20 ¼ 12:6 kJ=mol; U

x30 ¼ 8:4 kJ=mol;

Ux40 ¼ 8:4 kJ=mol; Arg: U

x10 ¼ 12:6 kJ=mol; U

x20 ¼

12:6 kJ=mol; Ux30 ¼ 12:6 kJ=mol; U

x40 ¼ 5:0 kJ=mol;

Val: Ux10 ¼ 12:6 kJ=mol; U

x20 ¼ 12:6 kJ=mol; U

x30 ¼

12:6 kJ=mol; Glu: Ux10 ¼ 12:6 kJ=mol; U

x20 ¼ 12:6 kJ=

mol;Ux30 ¼ 0:8 kJ=mol:Hydrogen bonding energy was

calculated based on Morze potential. Dissociation

energy of the hydrogen bond is taken to be 6.3 kJ/mol.

A rigid valence scheme of the molecule was assumed,

namely, the searches were made only on torsion angles.

The conformational state of each amino acid residue

is characterized by backbone w; c and side chain x1,

x2, x3,… dihedral angles. The terms ‘conformational

state’ or ‘conformation’ used in the following analysis

will always imply exact quantitative characteristics of

residue or fragment geometry. For a stable confor-

mation, the w and c dihedral angles are located in the

low energy regions Rðw;c ¼ 2180 to 08Þ; Bðw ¼

2180 to 08; c ¼ 0–1808Þ; Lðw;c ¼ 0–1808Þ; Pðw ¼

0–180; c ¼ 2180 to 08Þ of the conformational map.

The notion ‘form of a residue’ to denote the above-

mentioned regions of its backbone dihedral angles was

introduced. Therefore the conformational state of each

aminoacid residue is conveniently described by Xij

where X is the backbone of a residue (R,B,L,P), and

ij ¼ 11…; 12…; 13…; 21… specify the positions of a

side chain (x1;x2; x3;…), the index «1» corresponds

to the x angle in the range from 08 to 1208, «2»

corresponds to the angle range from 1208 to21208 and

«3»—from 2120 to 08. The nomenclature and

conventions adopted are those recommended by

IUPAC-IUB [16].

The combination of the backbone forms of a residue

in a given aminoacid sequence will specify the

backbone forms of a fragment. So, all backbone

forms of a dipeptide can be classified into two types,

folded and extended. The folded type of backbone of

the dipeptide is represented by RR, RB, LL, LP, PR,

PB, BL and BP forms and the extended forms by BB,

BR, RL, RP, PL, PB, LB and LR forms. Forms,

belonging to a particular type have an analogous

peptide chain contour and a similar mutual arrange-

ment of backbones and side chains and should exhibit

similar medium-range interaction potentialities. The

number of possible conformations within each form

depends on the nature of a residue. It should be noted

that all backbone forms and peptide chain types are

initially presumed to be equivalent. But the application

of the theoretical conformational analysis for investi-

gation peptide molecules colloids with the problems of

multiplicity of calculated conformations. In fact,

the possible starting conformations for the peptide

with n-aminoacid residues may be 10n. In order to

abridge the number of conformations we used the

approach to peptide structure calculation based on the

fragmental analysis with using the universal sets of

low-energy conformation states of the free aminoacids

and tested on numerous peptides, as for example in

Refs. [17–21].

3. Results and discussion

The scheme of calculation and stages of calcu-

lation are illustrated in Fig. 1. As can be seen from this

figure, the molecule was divided into fragments under

consideration of its biological activity. The results of

the investigation of the structure of Met-enkephalin

N.A. Akhmedov et al. / Journal of Molecular Structure 646 (2003) 75–8076

and adrenorphin were presented in our previous works

[20,21], where it was be shown that the spatial

structure of the Met-enkephalin and adrenorphine

may be described by five and 13 types of the peptide

backbone, respectively. The starting conformations of

BAM-12P were constructed from these stable confor-

mations of the adrenorphin, four forms of

monopeptide Gly and the low-energy states of the

Arg10-Glu12 tripeptide fragment, which was carried

preliminary on the basis of the conforming monopep-

tide states. Thus, in the first stage a number of

structures of BAM-12P to be analysed amounted to

600. The dihedral angles of the adrenorphine’s part of

the molecule was keep fixed and only the Gly9-Glu12

was varied. From the examined variants of BAM-12P

molecule 400 were steric prohibited: the relative

energy of others was delivered from 0 to 168 kJ/mol.

Then a restricted number of conformations with

relative energies in a sufficiently wide interval of 0–

65 kJ/mol was selected for further consideration. In

these conformations the dihedral angles of the main

and side chains of the all aminoacid residues were

varied, and the side chain positions were specified. The

best optimal conformations of molecule the energy of

which were not more than 30 kJ/mol are represented in

Table 1. The right side of Table 1 contains the relative

energies and separate energy contributions. As may be

inferred from this table, all preferable structures of

BAM-12P are composed of combination of eighth low

energy conformations of adrenorphine, two forms of

monopeptide Gly9 and two forms of Arg10-Glu12

tripeptide. The central fragment Arg7-Val8-Gly9 is

labile as compared to the previous and subsequent

parts of the given molecule. The geometrical par-

ameters (in degrees) for the low energy conformations

of the different forms of the BAM-12P are represented

in Table 2. The atomic model of their spatial structure

is shown in Fig. 2(a)–(d) correspond to the structures

with the relative energies 0, 7.6, 9.2 and 17.2 kJ/mol,

respectively. As seen from a figure, the backbone

forms of the studied molecule drawn in the most visual

projections, derivate the flexural structures resulting in

to steric approach of the residues, removed along a

chain. It is reached due to mobility of the central

segment of the molecule, connected the conformation-

ally rigid parts of the molecule. The high density of

packaging of a polypeptide chain of the most

preferential structures is accompanied by installation

of the numerous hydrogen bonds.

The global conformation of this molecule is B211-

PRR21B332R2222R3222R2BL22RR32 (Fig.2 (a)). Here

two helical segments, revealed on the fragments Tyr1-

Met5 and Arg6-Arg10, respectively, are removed

regarding to one another due to extended form of

connected them dipeptide fragment Met5-Arg6 and

derivate the reverse turn of the polypeptide chain. The

folded form of majority of aminoacid residues in this

structure promotes the realization of the abundance

dispersion contacts of backbone chain atoms. The

favourable contacts between the atoms of the remote

parts of the molecule are also realized in this structure

due to existence of the abovementioned turn on central

fragment of peptide. On this cause the global

Fig. 1. The scheme of calculation of the BAM-12P molecule.

Table 1

The energies (kJ/mol) of favourable conformations of BAM-12P peptide molecule

Conformation Erel Env Eel Etors

1 B211 P R R21 B332 R2222 R3222 R2 B L22 R R32 0 2173.9 35.7 26.9

2 B131 B P B21 B212 B1222 B2222 R2 B L22 R R32 7.6 2172.6 43.3 25.2

3 B132 P R B33 B222 B1222 B2222 R2 B L22 R R32 9.2 2168.8 42.4 24.4

4 B212 B P R21 R212 B1222 B2222 R2 B L22 R R32 17.2 2181.9 49.6 38.2

5 B132 P R B33 B222 B1222 B2222 B2 R B21 R R32 22.3 2168.0 38.2 40.3

6 B131 B P B21 B212 B1222 B2222 B2 R B21 R R32 25.6 2167.2 47.0 33.6

7 B131 B P B21 B212 B1222 B2222 B2 B L22 R R32 27.3 2166.7 45.8 37.0

8 B131 B P B21 B212 B1222 R2222 B2 B L22 R R32 28.6 2179.3 66.8 29.4

9 B132 P R B33 B222 B1222 R2222 B2 B B21 R R32 29.4 2174.7 62.2 30.2

N.A. Akhmedov et al. / Journal of Molecular Structure 646 (2003) 75–80 77

conformation is compact, that makes it preferential

both on nonvalent and on electrostatic interactions.

Our calculations showed that in the majority low

energy structures the side chains of Tyr1, Phe4

have conformational mobility because of its

localization on surface of the molecule. It is

substantiated by physiological expediency: such

mobility of the aromatic rings is probably necess-

ary for complementary binding with the specific

receptors. The side chains of the residues Arg6,

Arg7, Arg10, Glu12, having the charged atom

groups oriented into the environment and interacted

neither one another nor with a backbone. Therefore

they are able to install the hydrogen bonds with the

solvent. In spite of presence of two glysine residues

in the Tyr1-Met5 fragment of the molecule,

corresponding to Met-enkephalin sequence, this

part of peptide molecule is not labile, it has the

particulate-folded structure.

Four types from five possible for Met-enkephalin

stable backbone forms [20] realized for fragment

Tyr1-Met5 at formation of spatial structure of BAM-

12P molecule. As it is visible from the represented

structures in Fig. 2(b)–(d) the fragment Tyr1-Met5

includes a turn of a chain with the residues Gly3 and

Phe4 in a center of turn with the approaching of the

atom groups NH Met5 and CO Gly2. These results are

in accordance with the available data of the

experimental and theoretical investigations of Met-

enkephalin [22–27], which introduce such structure

with b-turn as predominant. It is possible to suppose,

that exactly the indicated turn of a chain is a major

factor in charge of opiate activity as in Met-

enkephalin, so in adrenorphine and BAM-12P.

As seen from Table 1, the contributions of the

nonvalent interactions change from (2166.7) up to

(2181.9) kJ/mol and electrostatic interactions in

the interval 35.7–66.8 kJ/mol. Therefore the value of

Table 2

The geometrical parameters (in degrees) of the BAM-12P peptide molecule for the low-energy conformations, belonging to the different forms

of the backbone

Residue No conformation

1 2 3 4

Tyr 1 2173 154 176 293 158 2179 167 158 179 259 140 2177

2177 75 0 50 298 0 59 295 178 2169 92 180

Gly 2 80 283 180 286 92 175 80 271 180 2151 79 172

Gly 3 267 242 2176 90 260 179 2161 270 178 80 279 2176

Phe 4 284 252 180 2139 148 2179 2137 63 179 294 243 2172

180 74 180 71 260 277 179 2113

Met 5 2148 126 174 273 109 2179 2155 146 2179 2135 269 2160

275 260 180 174 58 2179 2167 177 2177 2171 60 2178

180 180 180 180

Arg 6 296 255 177 2164 170 170 2144 170 170 2159 170 170

264 180 179 64 2175 2178 64 2175 2178 64 2175 2178

180 179 179 179

Arg 7 2131 134 180 2117 126 2176 2117 126 2176 2117 126 2176

2177 177 180 2179 178 180 2179 178 180 2179 178 180

180 180 180 180

Val 8 2100 260 2172 2109 260 179 2109 260 2174 2109 260 2175

180 2179 179 179

Gly 9 271 101 2178 268 117 178 267 118 178 268 117 179

Arg 10 58 84 175 58 84 175 58 84 175 58 84 175

2177 180 178 2117 180 178 2177 180 178 2177 180 178

180 180 180 180

Pro 11 260 253 180 260 253 180 260 253 180 260 253 180

Glu 12 2103 263 – 2103 263 – 2103 263 – 2103 263 –

260 180 90 260 180 90 260 180 90 260 180 90

Urel (kJ/mol) 0 7.6 9.2 17.2

Comment: The values of dihedral angles in the table are in the following order: f; c; v; x1; x 2,….

N.A. Akhmedov et al. / Journal of Molecular Structure 646 (2003) 75–8078

total conformational energy for represented confor-

mations of BAM-12P can undergo noticeable change

under influence of a solvent or contacts to the receptors.

4. Conclusion

Because of a great sensitivity of a position of the

conformational equilibrium of a molecule to the nature

of an environment and of an intermolecular inter-

actions all represented conformations of BAM-12P are

potentially physiologically active and should be

allowed in the subsequent analysis of the structure-

functional organization of this molecule. Thus, theor-

etical conformational analysis of BAM-12P molecule

lead to the such its structural organization, which do

not exclude the realization of many various functions

demanding strict specific interaction with the different

receptors. The knowledge of the active conformations

of a given peptide is a major step towards under-

standing its biological functions. There is an

impression, that the identical conformation of Met-

enkephalin, adrenorphine and BAM-12P is responsible

for the realization of their common functions, and the

quantitative effect and its duration depends on length of

the subsequent peptide chain. The received data also

will allow to plan the producion of pseudoanalogues of

BAM-12P with modified properties useful in therapy

and medical diagnostic.

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