Download - Conformational analysis of human growth hormone [6-13] peptide analogues

Int. J. Prpridr Protein R ~ J . 48, I Y Y 6 . 1- I 1 Printed in UK all ri,qhts rcsrrrrd

PEPTIDE & PROTEIN RESEARCH ISSN 0367PXj77

Conformational analysis of human growth hormone [ 6-13] peptide analogues

KERRY A. HltiGINS. PHILIP E. THOMPSON and MILTON T.W. HEARN

Centre for Bioprocess Technology. Deptrrtineiit of' Biocheinistrj. anti Mo~eciilur Biolog?,, Monash University, Clayton, C'ictoriu, Aiisfrulici

Received 28 March, revised 30 August. accepted for publication 22 October 1995

The conformational analysis of a series of ten hGH[6-131 peptide analogues is reported. As part of our earlier studies, the r-aminosuccinimide modified fragment Asu1'-hGH[6-l 31 has previously been identified as a potentiator of insulin activity in intravenous insulin tolerance tests, and various analogues have been subsequently designed, synthesised and employed to acquire structure-activity data. These studies have lead to the conclusion that the conformational characteristics a t the C-terminus of each of the active peptide analogues is important to the biological activity. In the present investigation, molecular dynamics and simulated annealing techniques have been used to examine the accessible conformational states of the C-terminal region of ten different hGH[6-13] peptide analogues. Of these six are active peptide analogues while the other four show no biological activity. Examination of the conformer groups identified using this molecular dynamics approach showed a common conformational motif for each of the active peptides. Q Munksgaard 1996.

Key words; computer modelling: molecular dynamics: peptide analogue: peptide conformation; simulated annealing

Discrete fragments of human growth hormone (hGH) show insulin-like activity. A number of previous investigations have concluded that the N-terminal peptide region of hGH was responsible for this activity ( 1-4). The r-aminosuccinimide modified fragment Asu"-hGH[6-13] (1) was subsequently identified as an in vivo potentiator of insulin activity with intravenous insulin tolerance tests and a modulator of glyco- gen synthesis in other in vitro bioassays ( 5 , 6). The observed activity makes this octapeptide and particularly the derived ~-lactam1'-hGH[6-l 31 analogues attractive lead compounds for the development of new therapeutic agents for potential use in type I1 diabetes treatments.

Previous investigations in this laboratory have sought to identify the mechanism by which this octapeptide and its analogues exert their biological effects. Various analogues of the a-aminosuccinimido- peptide (1) have been designed, synthesised and subsequently used to accumulate detailed structure- activity data (1-4). It has been found that within the N-terminal region of these peptide analogues the chemical composition of the amino acid side chains represents the dominant determinant of activity. In contrast, these studies have shown that it is the conformational characteristics of the C-terminal

region of these peptide analogues rather than their chemical compositional features per se that appear to be far more important for the expression of the biological activity by this sequence region. In particular. substitution of the metabolically unstable Am"- Asn" segment by a range of other structural entities with similar conformational features has lead to the development of peptide analogues with similar or enhanced potencies in the intravenous insulin tolerance test (IVITT) in the both the normal and the diabetic rodent animal models (6-1 1). Figure 1 illustrates examples of these active peptide analogues. It has previously been suggested that a Type 11' p-turn conformation may occur in this Asu"-AsnL2 region of the hGH[6-131 derived peptide (1). In order for our analogue design programme to develop further, it was important to examine in detail the nature of the conformational constraints imposed by the amino acid residues at positions Aaa( 11) and Aaa( 12) in peptide (1) and its active variants.

Molecular dynamic/simulated annealing techniques are increasingly being applied as an integral compon- ent of the rational design of peptide analogues and peptidomimetics (12, 13). The ruison d'ttre of many previous molecular dynamics/simulated annealing studies with peptides and proteins has been the ulti-

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K.A. Higgins et al.

Peptide (7): Pepode (8): hGH 16-131:

Leu-Ser-ArgLeu-Phe- X- Y- AlaNH2

Peptide (1): Pepbde (2):

Pepllde (3) Peptide (4)

Peptide (6): Peptide (5):

FIGURE 1 The amino acid sequence of the insulin potentiating peptide, hGH(6-13), and each of the active XY peptide fragments where peptide 1 incorporates the Am"-Am", peptide 2 the 3-amino-y- lactam"-Gly", peptide 3 the 4-arnino-y-la~tam"-Gly'~, peptide 4 the ~-Ala"-Pro'', peptide 5 the D-Pro"-Asn'' and peptide 6 the /l-Asp"-Asn" residues. The d2, $', d3 and $3 dihedral angles for each of the peptides are illustrated.

mate aim to identify the conformation at the global minimium. Our aim in this study was to identify the conformations available to each of the peptide fragments. In high-temperature molecular dynamics simu- lations, the available thermal energy is used to climb and cross-conformational energy barriers. Consequently, molecular dynamics (MD) provides information into accessible conformational states of the molecule. Previously M D procedures have been used with a variety of small peptide systems such as vasopressin ( 14) and gonadotropin-releasing hormone (14, 15) to find low-energy conformational states across energy barriers that would be inaccess- ible to classical minimisation strategies. In the investigations described in this paper, a similar molecular dynamics/simulated annealing approach has been used to examine the conformations available to a series of ten peptides of the general sequence Leu- Ser-Arg-Leu-Phe-X-Y-AlaNH, in which the X-Y residues have been substituted. Both active (Fig. 1) and the inactive (Fig. 2) peptides were investigated. Our primary interest in this study was to examine the conformations available to the C-terminal region of the peptide, and in particular, the X-Y fragment.

2

Peptlde (9) Peptlde (10)

FIGURE 2 The XY sequence for each of the inactive peptides investigated. Peptide 7 incorporates the L-Pro"-GlylZ, peptide 8 the ~-Asp"- Am", peptide 9 the L-Aka"-Pro" and peptide 10 the /&Asp"- Asn" residues. The &, $*, d3 and $3 dihedral angles for each of the peptides are illustrated.

METHODS In this investigation, a total of ten peptides were examined with the general sequence of Leu-Ser-Arg- Leu-Phe-X-Y-AlaNH,, where X-Y is: ( 1) Asu"- Asn", (2) 3-amino-y-lactam"-Gly'z, (3) 4-amino-y- lactam"-Gly'2, (4) D-A1a1l-Prol2, ( 5 ) D-Pro'l-Asn'Z, (6) P-Ala"-Asn12, (7) L-Pro"-Gly'2, (8) ~-Aspl l - Asn',, (9) L-Ala"-Pro", (10) p-Asp"-Asnl2. Each of the peptides 1-6 is an active hypoglycaemic analogue of the hGH[6-13] peptide, while biological testing has shown that peptides 7-10 are inactive.

Molecular dynamics Energy calculations on the peptide series employed the CVFF potential force field. Using this force field, parameter assignments (bonds, valence angles, tor- sion angles, out-of-planes and 0-0 cross-terms) were made automatically (i.e. it was not necessary to make estimates of parameters: all were available as pre- determined). No Morse functions or cross-terms were used. An initial geometry for each peptide analogue was generated by building each of the sequences as an extended conformation using standard amino acid bond lengths, angles and sidechain dihedrals. Solvent was not included and all the amino acids were defined to be in their neutral states. All generated conformers were examined with the criterion employed that there was no high-energy cutoff, as higher-energy conformations may be stabilised on binding to the receptor. Peptide bonds were forced to remain in the trans configuration by applying a torsional restraint of 10 kcal/mol rad2 during the calculations.

Energy minimisation of the starting structures was achieved using the steepest descents minimisation algorithm ( 16) followed by conjugate gradients minimisation (16) until a maximum derivative of

hGH[6-13] peptide analogues

approach followed in this investigation and to high- light the observed changes, these dihedral angles are plotted as a function of time (ps) in Fig. 3. Since conformers were collected at 1 ps intervals, the dihedral angles for each of the derived 100 conformers are given. The d2 dihedral angle shows two preferred angle ranges. The majority of conformers fall in the 42 dihedral angle range of - 70 to - 180", whilst a second smaller group appears approximately 30 ps into the trajectory and shows a 42 dihedral angle range of 40-70". The t+b2 dihedral angle shows a single angle range from - 100 to - 140°, which is consistent with the restriction imposed on this bond by the a- aminosuccinimide ring structure. For the 43 dihedral angle two distinct ranges of -70 to -130" and 40-90" were observed. As there is only a single transition observed between the two conformer groups, this result indicates that there exists a consid- erable energy barrier to the interconversion. The $3

dihedral ranges (Fig. 3b) are 70-130" and -30 to - 80" and transitions between the conformers are

0.1 kcal/mol was reached. The MD run involved an initial equilibration at 1000 K for 1000 steps of 1 fs. Data were collected from a subsequent 1OOps MD run at 1000 K. This approach resulted in the genera- tion of 100000 conformers (dynamics steps of l fs) for each peptide analogue. Instantaneous dynamics structures were collected at 1 ps intervals giving a total of 100 conformers for each peptide analogue. For each of the conformers the temperature was then gradually reduced to 300 K over a period of 30 ps. Equilibration at 300 K was achieved using a 10 ps run of MD. For each of the peptide analogues 100 conformers were generated at 300 K, after which the conformer groups were minimised using the steepest descents method until the maximum derivative was less than 0.1 kcal/mol. This procedure was followed by quasi-Raphson-Newton minimisation until a maximum derivative of lo-' kcal/mol was obtained. Minimisation had the effect of merely tightening the dihedral angle ranges, since it was found to be most efficient to minimise using the above protocol prior to sorting the conformers into groups.

Analysis of conformers Conformers were sorted based on the measured dihedral angles for those bonds in which some degree of freedom was possible. Along the backbone for peptides 1-5 and 7-9 a total of six flexible bonds are examined, while peptide 6 and 10 both incorporate the additional flexible bond, labelled p (Figs. 1 and 2). As our primary concern was the X-Y fragment, and conformations available to this moeity, the initial sorting of peptides was consequently based on the dihedral angles d2, t+b2, 43 and t+b3. Sorting of the dihedral angles resulted in the identification of a series of conformer families for each of the peptides. For a particular conformer group an average dihedral angle is reported along with the dihedral angle range. Only groups which include greater than 5% of the total conformer population were regarded as 'stable' conformations (17). On completion of the grouping procedure, other backbone and sidechain dihedral angles were examined, as was the likelihood of H bonding. A hydrogen bond (H bond) was considered highly probable if the prFton-acceptor distance did not exceed 0.25 nm (2.5 A) and the proton-acceptor angle was larger (1 8) than 135".

RESULTS AND DISCUSSION

Peptide 1 The structure of peptide 1 is shown in Fig. 1. High- temperature molecular dynamics of this peptide was followed by annealing, and finally energy minimisation, resulting in the collection of 100 conformers. The first point of interest evident from the analysis of these conformers relates the nature of the dihedral angles 42, t+b2, q53 and t,hg. To illustrate the general

0 20 40 60 80

Trajectory time (psec)

b 1801

- 120

2 F 60

0 , o

-60

m

0 m - 2 - m r O -120

-180 1 0 20 40 60 80


FIGURE 3 The dihedral angles (a) d2 (+), GZ (0) and (b) d3 (a), c1/3 (+) as a function of the trajectory time (ps) for peptide 1. Each of the data points represents a single conformer which were collected at 1 ps intervals.

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K.A. Higgins et a1

relatively frequent. Conformers were grouped based on dihedral angle values, and as a consequence nine separate conformer families were identified.

Table 1 provides a list of the 42, t,b2, 43 and t,b3 dihedral angle values for each of the conformer groups identified, along with the proportion of the total conformer population showing a particular conformation. Seven of the nine conformers identified

TABLE 1

Conformer families arising from thr niolrciilor driiiimics mu!,, the Asu-Asn resitliirs of prptiili, I

Conformer Dihedral angles ( ) ''4, conformer family

9 2 $ 2 4 3 $3

6

1

1

26

a Each dihedral angle value represents the average. and the number in parentheses indicates the range 01' angles abailable for each of these dihedrals.

a Each dihedral angle value represents the average. and the number in parentheses indicates the range 01' angles available for each of these dihedrals.

for peptide 1 show an extended conformation. The other two (conformer groups 7 and 8) show a Type 11' 8-turn conformation based on the criteria of Lewis et (11. (19) and Capasso et al. (20). In addition each of the conformers making up the conformer group 7 fulfil the criteria (of distance and angle) for a H bond between the Phe CO and the Ala NH. In the Type 11' 8-turn conformation, a H bond distance consistent with a 1 +4 H bond is observed.

These findings with this peptide 1 are consistent with earlier X-ray crystallographic, circular dichroism and two-dimensional NMR studies (20-23) of CI- aminosuccinimide containing peptides which suggested 42 values of 55 and - 115", and t,b2 values limited to -120^.

Peptick 2 For peptide 2 conformers arising from the dynamics trajectory were treated in an identical manner to that discussed above for peptide 1. Initially the 42, t,b2, b3 and t,b3 dihedral angles were investigated. For the 42 dihedral angle two conformer groups were identified; one with a angle range of -80 to - 180", and the second with angles ranging from 30 to 70". The t,b2 angle was restricted due to the 3-y-lactam ring structure with a single range of - 100 to - 140" observed. For the 43 dihedral angle, two conformer groups were identified with dihedral angles in the range 90-120" and -70 to - 110'. Transitions at the & dihedral were more frequent with this peptide than observed for peptide 1. For the $3 dihedral angle two main conformer groups were identified with angle ranges of -90 to -40' and 50-90".

Sorting the conformers resulted in the identification of five separate conformer groups. Although the d 3 and t,b3 dihedral angles undergo a significant number of changes over the trajectory period, they effectively only move between two angle ranges. It appears from the results that relative to peptide 1, the energy barrier to the conversion is small, and may be attributed to the change in sidechain group from the Asn12 in peptide 1 to a Gly12 in peptide 2. Of the five conformer groups identified, four show extended structures whilst the conformer family 5 , fulfils the criteria for a Type 11' 8-turn, including the appropriate geometry and distance for a 1-+4 H bond. There has been some ambiguity in the literature as to whether a Type 11' 13-turn is the favoured conformation for the 3-y-lactam moiety (24-27). The present study has provided further supportive evidence that a range of conformations are possible with peptide analogues containing the 3-y-lactam moiety with the Type 11' 13-turn conformation making up of one of these groups.

Peptide 3 The structure of peptide 3, which incorporates the novel 4-amino-~-lactam" residue ( S ) , is given in

4

hGH [ 6-1 31 peptide analogues

TABLE 3 Conformer families arising from the molecular dynamics study of

the 4-amino-y-lactam-Gly residues of peptide 3

Conformer Dihedral angles (") ?h conformer family

4 2 *2 4 3 *3

a Each dihedral angle value represents the average, and the number in parentheses indicates the range of angles available for each of these dihedrals.

Fig. 1. The observed ranges for the d 2 ) $ 2 , 43 and $3

dihedral angles were very similar to those observed for peptides 1 and 2. The q52 bond shows dihedral ranges of 40-80", and -70 to - 160", with the latter conformer group being the most populated. The $2

dihedral angle shows a single range of - 80 to - 160". This range is larger than that observed for the other two peptides and can be attributed to the neighboring ring bond which in this instance does not create as restricted a peptide bond as in peptides 1 and 2. The q53 and $3 bonds each show two conformer groups both centred at approximately 80". However, both conformer groups also show a large number of transitions between conformers in much the same way as peptide 2. Division of the conformers into groups resulted in the identification of four different conformer families (Table 3). Conformer family 4 fulfils the criteria for a Type 11' p-turn.

Peptide 4 Designed as a Type 11' p-turn analogue, peptide 4 represents a significant change compared with peptides 1-3 with respect to chemical composition of the X-Y fragment (Fig. 1 ). Conformers generated using the molecular dynamics method are divided into groups based on the procedure discussed above. The 42, $ 2 , d3 and $3 dihedral angles were examined initially. Each of the dihedrals is shown as a function of the trajectory time (Fig. 4) in order to illustrate the differences with this peptide 4 compared with peptides 1-3. The $2 dihedral angle from the D-Ala residue shows a range of angles from 60 to -130". After the minimisation the $ 2 dihedral shows angle ranges of - 60 to - 130" and a second group (showing a very small number of conformers) with angles of 40-60". The 43 bond in this case forms part of the Pro ring and as expected shows a single, limited dihedral range of -40 to -90". At the ~ + b ~ dihedral

-180 J

0 20 40 60 80


- 120 '"1 v)

2 p 60 9 G O 4

{ -60

I:

m

- 0

lY -120

-180 ' 0 20 40 60 80


FIGURE 4 The dihedral angles (a) Qz ( O ) , $2 (+) and (b) b3 (C), $3 (+)as a function of the trajectory time (ps) for peptide 4. Each data point represents a single conformer which were collected at 1 ps intervals.

angle, two groups of dihedral angles are identified and these range from 50 to 90" and - 15 to - 50".

Table 4 shows that for peptide 4 only three separate families of conformers were identified, and of these, the conformer family 1 fulfils the criteria for a Type 11'

TABLE 4 Peptide conformer , furdies arising from the molecular &unrics

study of the DAla-Pro residues of peptide 4

Conformer Dihedral angles (") [YO conformer family

4 2 *2 4 3 *3

1 77 -113 -75 -35 2 6

76 59 2

3 120 53 -78 82 7

(+15)" (k20) ( i 2 0 ) (1-15) 109 -89 -73

( + I 5 1 (+20) (+lo) (+20)

(+5) ( + 5 ) ( 1 5 ) (*lo)


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K.A. Higgins et al.

/I-turn. For the Type 11' /?-turn conformation a 1 +4 H bond is indicated. The derived conformers for this peptide differ significantly to those identified for peptides 1-3 in all except one instance, as a single conformer family which is common for all three peptide analogues is evident represented by the Type 11' /?-turn.

Peptide 5 The structure for peptide 5 is given in Fig. 1. Analysis of the data revealed that the d 2 bond was restricted by the Pro ring and consequently only a single dihedral range of 50-90" was observed. The I,h2 bond showed two angle ranges, one at 20-60' and the second at - 70 to - 130". The majority of conformers fall into the second range. The 4 , bond shows a dihedral range from -60 to - 150'. Two separate conformer dihedral angle ranges were observed for the 4b3 bond from 60 to 120" and -25 to -50'. Grouping of the conformers resulted in the identification of five separate families of which two fulfil the criteria for a Type 11' 0-turn (conformer families 3 and 4). Neither of the Type It' b-turn conformations show the appropriate geometry to indicate the presence of a 1-4 H bond.

Peptide 6 The structure for peptide 6 is shown in Fig. 1. In the case of peptide 6 analysis of the conformers available to the XY fragment is complicated by the additional flexible bond labelledp. The 42, p, \clz, 4, and I,h3 dihedral angles were used to divide the conformers into groups. For the q52 bond, angles range from 90 to 150", and -80 to - 150', while similar ranges were also observed for the p and ~2 dihedral angles. Owing to thep bond, a greater degree of flexibility was expected at the 42 and I,h2 bonds than was

TABLE .5 Conformer fumilies arising from tile rnolecukur dwurnic's st i idi . qf

the D-Pro-Asn residitrs of peptide 5

Conformer Dihedral angles ( ) 'YO conformer family

4* $2 $ 3 $ 3

a The values in parentheses indicate the range of dihedral angles available to a particular conformer group dihedral.

6

observed for each of the other peptides. This greater flexibility was manifested as a larger number of transitions between the preferred conformations. For the 43 dihedral an angle range of 70-100" and -80 to - 160" was observed, while for I,h3 the dihedral angle ranges observed were 40-120", with only a very small grouping showing ranges of - 60 to - 90".

On completion of the grouping procedure a total of six different families of conformers were identified for peptide 6 (Table 6). Of the six families identified, three give rise to a turn conformation (families 1, 2 and 6) while the remaining three families are essen- tially represent linear or extended structures (Fig. 5 ) . Owing to the additional bond p, it was not possible to use the standard dihedral angle values for identification of the Type 11' 0-turn. Superimposition of each of the identified 'turn' conformers on a standard Type 11' fi-turn, resulted in an RMS deviation in each case of < 1.1 A When the same superimposition of the Type 11' 0-turn conformers were undertaken for the peptides 1-5, in !ome cases the RMS deviation was greater than 1.6 A.

For peptide 6 none of the identified turn conformations shows as tight a turn as the Type 11' [&turn. Consequently, there was no possibility of a 1 4 4 H bond. However, the presence of a stabilising intra- molecular H bond was indicated as occurring between the Ala NH and 0-Ala CO for conformer family 2. Based on the measured RMS deviation between the peptide 6 'turn' conformers and the standard Type 11' 0-turn, i t can be proposed that three of the six 0- Ala-Asn peptide conformer families are equivalent conformationally to the Type 11' /?-turn.

Pepticle 7 Each of the peptides 7-10 (Fig. 2) have been shown to be inactive in the intravenous insulin tolerance tests and other biochemical bioassays (4, 5) . For peptide 7, the XY fragment consists of a L-Pro'l- Gly" residue pair. Analysis of the conformer sets obtained from the molecular dynamics study, resulted in the identification of five separate conformer families (Table 7 ) . As expected, owing to the pyrrolidine ring of the L-Pro residue, the range of angles available to the 42 dihedral was limited to -40 to -80". Consequently, this peptide shows conformer groups in common with most of those identified for peptides 1-3 with the important exception of the Type II' /?- turn conformer family. Peptide 7 shares no conformer groups with peptides 4 and 5.

Peptide 8 Peptide 8 incorporates the Asp"-Asn" residues at the XY position. Studies of the insulin potentiating ability of peptide fragments derived from the hGH protein, initially identified the native hGH( 6-1 3) fragment as responsible for the biological response. This peptide incorporates the Asp" and Asn12 res-

hGH[ 6-1 31 peptide analogues

TABLE 6 Conformer families arising from the molecular dynamics study of the fi-Ala-Asn residues of peptide 6

Conformer family Dihedral angles (") %I conformer

42 *2 P 4 3 $3

~~ ~


FIGURE 5 Conformers identified for the fi-Ala"-Asn12 fragment of peptide 6. The numbering corresponds to each of the groups defined in Table 6.

idues. However, further studies of the insulin potentiating capability showed that this was in fact not an active peptide fragment (4, 5). Analysis of the trajectory derived from the molecular dynamics study of peptide 8 resulted in the identification of five conformer families (Table 8). For this peptide there is a great deal of similarity between the identified peptide families and those observed for peptides 1-3. The

greatest difference compared with these other peptides arises for the $2 dihedral which shows angle ranges of - 30" to - 90", and 60-90". None of the identified conformer groups of peptide 8 fulfils the criteria for a Type 11' b-turn. Family 5 comes closest in terms of these criteria; however, the t,h2 dihedral angle results in a significant distortion from any turn conformation.

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K.A. Higgins et cil.

TABLE 7 Conformer furnilies arising from rlw riiolrcirlur c l i ~ r i i r r i i k , ~ s r i i c l j . of'

the L-Pro-Glj. rrsidirc~s of pc.(irirlt~ 7

Conformer Dihedral angles ( ) (1, conformer . family

4 2 iz 6 3 i 3

a Each dihedral angle value represents the average. and the number in parentheses indicates the range of angles available for each of these dihedrals.

Conformer Dihedral angles ( ) 'I , j conformer family

6 2 $ 2 6 3 $ 3

a Each dihedral angle value represents the average. and the number in parentheses indicates the range of angles available for each of these dihedrals.

Peptide 9 Peptide 9 incorporates the L-Alall-Pro" pair, and so differs from peptide 5 only with respect to chirality at the C" carbon but in all other respects is chemically equivalent. Only three conformer families were identified for this peptide (Table 9). The dihedral angle ranges observed include for the $ 2 dihedral, a single angle range of -70 to - 15Y, which in itself effectively rules out the possibility of a Type 11' /?-turn conformation occurring. A comparison of this peptide with peptide 5 particularly emphasises the significance of conformation for the biological activity of these peptide analogues. The MD study illustrates how important the chirality at a single C atom can be to

8

TABLE 9 Corffoniirr groups uri.\ing,fioni the molecular dynamics stud11 of the

i.-Alir-Pro jkagtnrnr in peptidr 9

Conformer Dihedral angles ( ' ) YO conformer famtlq

6 2 $ 2 4 3 $3

(i-10)" (* lo) (k5) (*I51

( i 1 5 ) (* IS)

(f20) (i20) ( + I S ) (k15)

1 -145 -65 -80 -80 5

2 -110 102 -64 -38 15

3 - 90 105 -76 88 71

a Each dihedral angle value represents the average, and thc number in parentheses indicatcs the range of angles available for each of these dihedrals.

the geometry and energy characteristics of the observed conformers.

Peptide 10 Peptide 10, like peptide 6, incorporates a /?-amino acid residue, as part of the Aaa"Aaa" fragment, and consequently it is necessary to include the additional flexible bond, labelled p. As was the case for peptide 6, this means that it is not feasible to use the standard dihedral angles as criteria for the identification of a Type 11' /)-turn. Figure 6 illustrates each of the conformers identified for this peptide and the dihedrals

3 4

FIGURE 6 Each of the conformers identified for peptide 10. The numbering corresponds to each of the groups identified in Table 10.

hGH [ 6- 1 31 peptide analogues

for each conformer group as given in Table 10. Figure 6 clearly shows that no turn conformation can be identified. Furthermore, there is no similarity between the identified conformers in peptide 6 and 10, even for the linear conformations. Peptide 10 shows a great deal of conformational flexibility. Based on the 5Yo abundance rule for a conformation (17), there are only four families identified for this peptide. However, these four families only represent a total of 34% of the complete conformer population. The remainder of the conformers form very small groups which have been considered for the purpose of this analysis as transitionary.

CONCLUSIONS

As noted previously (7-lo), the conformational features of the Aaa'lAaa12 region in the hGH(6-13) peptide analogues appear to represent a particularly important parameter in controlling whether or not these peptides exhibit hypoglycaemic biological activity. Peptides 1-3 show a great deal of conformational similarity with many of the conformer families identified for each peptide showing effectively the same dihedral angles. This result is consistent with the similar structures of each of these peptides. The Aaa11Aaa12 residue peptides 4 and 5, however, have

TABLE 10 Conformer groups arising fFom the molecular dynamics study of the p-Asp-Am residues of peptide 10

Conformer family Dihedral angles (") YO conformer

42 $2 P 4 3 $3

1 - 130 - 170 - 60 - 88 84 5

2 - 102 - 170 125 - 90 78 9

3 - 70 - 60 -113 -91 81 12

4 81 - 161 123 -83 72 8

( f 3 0 Y (* 10) (k 15) (k 10) i+ 15)

( k 2 5 ) (k 10) (k25) (k 10) (f 10)

i+20) (+ 15) (k25) (k20) (k 10)

(+25) (+ 15) (k20) (f 10) i k 2 5 )


FIGURE 7 Each of the Type 11' p-turn conformations for peptides 1-6. The numbering corresponds to the peptide number.

9

K.A. Higgins et al.

very little in common chemically or conformationally with the Aaa"Aaa" residues in peptides 1-3. Nevertheless, there is one single conformation common to all the active peptides examined, and this is represented by the Type 11' b-turn conformation (Fig. 7). In addition, the conformations available to the four inactive peptides were examined using the same molecular dynamics and analysis methods. For each of these peptides the Type 11' p-turn conformation was absent.

Based on the above MI> analysis the conclusion can thus be reached from this study that a Type 11' p-turn conformation at the carboxy terminal region of hGH(6-13) analogues may be important for the expression of biological activity with these peptides. It is thus of relevance that all the peptide analogues which exhibit bioactivity manifest this conformation as a common feature within the total repetoire of their conformational space. Previous studies, notably from the research groups of Capasso et ul. (20, 21 ) and Schon et al. (28) have provided evidence from X-ray crystallographic procedures and chemical modification methods on the presence of a Type 11' p-turn with model Asu-containing di- and tri-peptides in the solid state. However, high-resolution two- dimensional NOESY NMR studies with this group of peptide analogues suggest that little secondary structure exists for most of these analogues and all of their L-a-amino acid analogues in DMSO-d, or phoshate-buffer solutions ( 29, 30), yet in hydro- phobic chemical environments (31) or at the receptor level they can be readily discriminated as peptides with important secondary structural and functional differences.

The formation of a-aminosuccinimides in polypeptides and proteins represents an important and relatively common event in their non-enzymatic degradation in vivo and in vitro via isomerisation, racemisation and deamidation ( 3 2 , 33). For example, a-aminosuccinimide intermediates have been implic- ated in the degradation of recombinant human growth hormone (34), hen egg white lysozyme ( 3 5 ) , a-crystallin B2 (36) and human epidermal growth factor (37). Besides their immediate relevance to the design of more potent hypoglycaemic analogues of Asu"-hGH[6-13], the present investigation thus also provides additional comparative data on the types of conformational constraints which might occur in the above and other polypeptides or proteins around the corresponding Asp-Aaa or Asn-Aaa peptide segment during the formation of r-aminosuccinimide intermediates which arise from the sequence dependent deamidation or deglycosylation of Asn residues and the isomerisation of Asp residues or alternatively during the preparation of larger polypeptides by semisynthetic methods.

10

ACKNOWLEDGMENTS

These investigations were supported by the National Health and Medical Research Council of Australia and the Australian Research Council.

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I . Chawla. R.K., Parka, J.S. & Rudman, D. (1983) Annu. Rev.

2. Wallis. M., (1988) in Cooke, B.A., King, R.J.B. & van der Molen, H.V., eds., Hormones and Their Action, Elsevier, Oxford. pp. 125-160

,Wed. 34, 519-547

3. Davidson, M.B.. (1987) Endocr. Rev. 8, 115-131 4. Frigeri. L.G., Teguh, C., Ling, N., Wolff, G.L. & Lewis, U.J.

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14, 485-490

Protein Rex 44, 568-581

(1996). Manuscript in preparation

J. Chrornafogr. 593, 103-117

hGH[6-I 31 peptide analogues

Address:

M. T. K? Hearn Centre for Bioprocess Technology Monash University Wellington Road Clayton, Victoria 3168 Australia Tel: 61 +3-905-3720 Fax: 61 +3-905-5882 Email: [email protected]

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