A Conformational Analysis ofLeucine Enkephalin as aFunction of pH

Mahalaxmi AburiPaul E. Smith

Department of Biochemistry,Kansas State University,

Manhattan,KS 66506-3702

Received 8 August 2001;accepted 18 January 2002

Published online 00 Month 2002 in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/bip.10158

Abstract: The conformations of Leu enkephalin in aqueous solution have been investigated as afunction of pH using molecular dynamics simulations. The simulations suggest the peptide backboneexists as a mixture of folded and unfolded forms (approximately 50% each) at neutral pH, but isalways unfolded at low or high pH. The folded form at neutral pH possesses a 23 5 hydrogen bondand a close head to tail separation. No significant intramolecular hydrogen bonding of the carbonyloxygens was observed in either the folded or unfolded forms of the peptide. Analysis of the Glycarbonyl oxygens and terminal groups indicated that, while the conformational population distri-bution of Leu enkephalin did vary noticeably as a function of pH, their hydration was essentiallyindependent of pH and in agreement with the available NMR data. Further study indicated that theunfolded state of the peptide was not random in nature and consisted of one major unfoldedbackbone arrangement stabilized by a persistent hydrophobic interaction between the side chains ofTyr and Leu. © 2002 Wiley Periodicals, Inc. Biopolymers 64: 177–188, 2002

Keywords: molecular dynamics; clustering, peptide hydration; backbone structure; protonationstates

INTRODUCTION

Enkephalins are endogenous opioid peptides withmorphine-like activity.1 These linear pentapeptides,composed of Tyr–Gly–Gly–Phe–Leu/Met, are impor-tant neurotransmitters having powerful analgesicproperties. Various studies have suggested the exis-tence of multiple receptors (�, �, �, and �), differen-tially located in the central nervous system, and withdifferent structural requirements for high affinity li-gand binding.2 Although the enkephalins have a highaffinity for the � receptors, they do bind to � recep-tors.3 This distracted binding affinity of the enkepha-lins, which appears to be based on the position of the

N-terminus and the two aromatic rings (R1 and R4),4

is largely attributed to the liberal conformational flex-ibility of the peptide.

The discovery of the enkephalins has promptedmany research groups to investigate the structural andfunctional aspects of these bioactive peptides with thepurpose of developing highly selective agonists andantagonists for use as pharmacological tools in opioidresearch. An excellent minireview of both experimen-tal and theoretical studies of the structural aspects ofthe enkephalins under different solvent conditions hasappeared recently.5 Many of the structural studieshave focused on nonaqueous environments that per-mit the characterization of a single conformation.

Correspondence to: Paul E. Smith, Department of Biochemis-try, 36 Willard Hall, Kansas State University, Manhattan, KS66506-3702; email: pesmith@ksu.edu

Contract grant sponsor: Kansas Agricultural ExperimentalStationBiopolymers, Vol. 64, 177–188 (2002)© 2002 Wiley Periodicals, Inc.

177

However, it is well known that the nature of thesolvent encasing the peptide is important, and isbelieved to have a sizable effect on the conforma-tions of the peptide.6 Hence, conformational studiesof peptides in nonaqueous solvents do not neces-sarily provide useful data concerning the generalconformational properties of the peptide. There-fore, as we are primarily concerned with the con-formations of the enkephalins under (almost) phys-iological conditions, we will restrict our discussionof the available experimental data to focus on theaqueous phase.

Numerous experimental studies have been madeconcerning the structure of Leu enkephalin in aqueoussolutions. Khaled et al. 7 studied the structure of bothenkephalins (Leu and Met) in different solvents using1H NMR, 13C NMR, uv, and CD spectroscopy, andproposed the existence of a concentration-dependentfolded monomeric form. Stimson et al.,8 who wereunable to reproduce these results using NMR, as-cribed the effect to the impurities present in the sam-ples and thus rejected the model. In 1986, Gupta et al.9

used nuclear Overhauser effects (NOE) measurementsin D2O to reveal the close proximity of the twoaromatic rings in space. This was also later shown tobe an artifact by Motta et al.10

Mounting evidence has indicated the presence of a24 5 intramolecular hydrogen-bonded �-turn confor-mation in water, as well as DMSO and reverse mi-celles, which is further stabilized by a head-to-tailinteraction between the charged amino and carboxy-late groups.11–13 However, later studies discountedthis arrangement as being improbable based on thefact that the hydration state of the carboxylate groupis identical at neutral and high pH.14,15 Further NMRstudies also indicate the abstinence of the Gly car-bonyl oxygens and amide protons from participatingin intramolecular hydrogen bonding, further suggest-ing the probability of an extended conformation inwater.15–17

Surprisingly, only one recent explicit solvent mo-lecular dynamics (MD) simulation has been per-formed on Leu enkephalin in aqueous solution.5 Theauthors used bent and linear conformations as prelim-inary structures and concluded that there was a pre-dilection for compact conformations in both the sim-ulations, as characterized by the formation of 2 3 5(bent simulation) and 3 3 5 (linear simulation) hy-drogen bonds. Unfortunately, the authors could notrule out the fact that another starting conformationwould yield different results.

In summary, no emphatic conclusion has beendrawn regarding the structural aspects of Leu en-kephalin, and therefore the structure of the enkepha-lins in aqueous solution remains an open question. In

this report we present the results obtained from longmultiple MD simulations, starting from folded andextended conformations of Leu enkephalin in aqueoussolvent at neutral, high, and low pH. A study of thehydration state of the Gly2 and Gly3 carbonyl groupsand the terminal groups is performed and comparedwith the results obtained from 1H, 14N, and 17O NMRstudies. Furthermore, we present a detailed clusteranalysis to determine the various conformationalstates sampled by the peptide.

METHODS

All the simulations were performed using the GROMOS96program and the 43A1 force field.18 Uncharged N- andC-terminal patches were built by reference to the unchargedside chains of lysine and glutamic acid, respectively. Threedifferent pH values (labeled low, neutral, and high) werestudied. The low, neutral and high pH simulations involvedN/C formal terminal charges of �/0, �/�, and 0/�, respec-tively. Deprotonation of the Tyr hydroxyl group at high pHwas not considered.

The initial conformation of the peptide was taken fromthe crystal structure of Leu enkephalin.19 The peptide wassolvated in a box of SPC water,20 with a minimum distanceof 1.0 nm from any peptide atom to the edge of the box.This resulted in approximately 1000 water moleculesaround the peptide and an initial box size of length 4.0 nm.The system was then treated with 50 steps of steepestdescent minimization. A twin range cutoff of 0.8 nm/1.4 nmwas employed and the nonbonded atom pair list was up-dated every 5 steps. Long range electrostatics were treatedusing the Poisson–Boltzmann reaction field approach,21

with a reaction field permittivity of 54.22

Molecular dynamics simulations were then performedusing a time step of 2 fs and SHAKE23 to constrain all bondlengths. Equilibration of the system involved a 50 ps sim-ulation at 300 K, followed by production runs of 10, 5, or2.5 ns. The simulations were performed under conditions ofconstant temperature (300 K) and constant pressure (1 atm)using the weak coupling approach.24 Coordinates weresaved every 1 ps for analysis. Details of the simulations aresummarized in Table I. Stochastic dynamics simulationswere also performed at 300 K with a dielectric constant of80 for a total of 50 ns using a time step of 0.5 fs and no bondconstraints. All non hydrogen friction coefficients were setto 2 ps�1 .

A cluster analysis was performed using previously de-scribed methods.25,26 To describe the procedure briefly: Allconformations were compared with each other and each pairof structures assigned a root mean square deviation(RMSD). The structure with the most neighbors (otherstructures with a RMSD within a set tolerance, d) definedthe central conformation of a cluster. All other structureswith a RMSD less than d from the central structure werethen included in the cluster. This procedure was then re-peated after removing the conformations already assigned to

178 Aburi and Smith

a cluster. We note that some previous cluster studies haveuse a value of d � 0.1 nm.26 However, if a histogram isgenerated from all the pair RMSD values, one observes amaximum in the distribution around 0.09 nm followed by aminimum around 0.14 nm (data not shown). Correspond-ingly, we have chosen to use a value of d � 0.14 nm todistinguish our clusters. Consequently, one generates lesswell-defined spatial clusters, but all dihedral values remainwithin the same local minima on their respective free energysurfaces. Clustering was performed based on comparisonsof the all-atom RMSD, the backbone RMSD, and the phar-macophore (N-terminus, Tyr ring, Phe ring, equallyweighted) RMSD. A heavy atom distance of 0.4 nm or lesswas used to define a hydrogen bond.

RESULTS

General Features of the Simulations

A summary of the simulations is presented in TablesI and II. It is clear from the peptide–solvent interac-tion energy for the different charge states that solva-tion of the C-terminus was substantially more favor-able than that of the N-terminus. During the 10 nssimulation of Leu enkephalin at neutral pH, the pep-tide changed from an initially extended conformation,to a folded conformation, and then back to an ex-tended conformation. The folded conformation wasstable for approximately 5 ns, and involved a 2 3 5hydrogen bond and a close arrangement of the termi-nal groups. The 5 ns simulations of Leu enkephalin atlow and high pH did not produce stable folded con-formations. This can be seen from the time historiesdisplayed in Figure 1. However, the simulations atlow and high pH did sample conformations with closecontacts between the terminal groups. In addition, thehigh pH simulation also sampled conformations,which possessed a 2 3 5 hydrogen bond. We con-clude that significant stability of the folded conforma-tion required the presence of both a charged N- and

C-terminus. Other possible hydrogen bonds (13 4, 24 5, 14 4, 14 5) were transient (�5%) in nature.

Ionic and hydrogen-bonding interactions were notthe only common features of the simulations. A closearrangement of the Tyr ring and the Leu side chainwas consistently observed (see Figure 1). This inter-action predominated during periods of the simulationswhen there were no intramolecular hydrogen bonds.Furthermore, at high pH the aromatic rings of Tyr andPhe adopted a stacked arrangement with a separationof less than 0.5 nm (see Table II). This appeared to bepossible only when the N-terminus was deprotonated.Presumably, as there was no penalty for disruption ofthe strong hydration shell of the amino terminus. Thesimulations suggest that, while there is no single con-formation for Leu enkephalin in solution, there aresignificant stabilizing interactions present that are de-pendent on the pH.

Two additional simulations were performed, start-ing from the folded conformation observed after 5 nsat neutral pH, to investigate whether the absence offolded conformations at low and high pH was a con-sequence of inadequate sampling. The results are alsopresented in Table I and Figure 2. The 2 3 5 hydro-gen bond was lost after 100 ps of the high pH simu-lation, while it remained almost intact at low pH.However, when the interaction was intact, fluctuationsin the hydrogen-bond distance were larger than thosefor the neutral pH simulation, suggesting decreasedstability of the folded arrangement. The additionalsimulations appear to confirm that the folded confor-mation is more stable at low pH than high pH, butclearly most stable at neutral pH.

Conformational Sampling

The time histories of the major dihedrals of Leuenkephalin at neutral pH are displayed in Figure 3.Multiple transitions were observed for the majority of

Table I Details of the Molecular Dynamics Simulationsa

pH Neutral Low High Low HighInitial structure Unfolded Unfolded Unfolded Folded FoldedPeptide atoms 57 58 56 58 56Peptide charge 0 �1 �1 �1 �1Water molecules 1015 1018 1016 1022 1020Simulation time (ns) 10 5 5 2.5 2.5Box volume (nm3) 31.902 31.973 32.737 32.096 32.127Total PE �42597 �42444 �42639 �42614 �42534Peptide PE �392 �168 45 �163 54Peptide–solvent PE �1080 �821 �995 �829 �1006Solvent PE �41429 �41628 �41531 �41769 �41696

a All simulations were performed in the NpT ensemble at 300 K and 1 atm. Potential energies (PE) in kJ/mol. The folded initial structurecorresponded to the structure obtained after 5 ns of the neutral pH simulation.

Conformational Analysis of Leu Enkephalin 179

dihedrals suggesting that an adequate degree of con-formational sampling was obtained. The time histo-ries also indicated that the transition from a folded to

an unfolded conformation occurred via rotationaround �4 after 5.5 ns. We have also performed astochastic dynamics simulation of Leu enkephalin at

FIGURE 1 Time histories for selected distances from the simulations at three different values ofpH. The 23 5 hydrogen bond corresponds to the distance between the Gly N and Leu C atoms. TheTyr–Leu distance was defined by the center of the aromatic ring to the C� of Leu. All simulationsstarted from an unfolded conformation.

Table II Average Distances (nm) from the Simulationsa

Distance

Neutral pH

Low pH High pHFolded Unfolded Average

N—C 0.48 1.01 0.73 1.00 1.20235 0.36 0.94 0.65 0.94 1.03134 0.78 0.89 0.83 0.79 1.03245 0.47 0.66 0.56 0.66 0.68144 0.70 0.62 0.66 0.58 0.60N—R1 0.48 0.51 0.49 0.49 0.49N—R4 1.04 1.09 1.08 0.98 0.70R1—R4 0.96 0.92 0.96 0.86 0.65R1—Leu 0.77 0.65 0.73 0.98 1.02

a Hydrogen-bond distances refer to the heavy atoms, or the carboxylate carbon for the acceptor of residue 5. Ring positions were definedby the center of the 6 carbon atoms. The C� atom was used to define the position of the Leu side chain. The folded and unfolded datacorrespond to averages over 1–5 and 6–10 ns of the neutral pH trajectory. All simulations started from an unfolded structure.

180 Aburi and Smith

neutral pH as a further test of the degree of samplingprovided during the explicit simulations. The simula-tion was performed with a crude representation of thesolvent (constant dielectric). Nearly all the possibleconformations of Leu enkephalin are sampled withthis representation, although not with the weightsexpected for an explicit solvent simulation. Probabil-ity histograms for the major dihedral angles werecreated from the explicit solvent MD and implicitsolvent SD data and are displayed in Figure 4. The �dihedrals possessed maxima at values of �120, �60,and 60°, the � dihedrals at �60° and 120°, and the dihedrals at �180°, �60°, and 60°. The Gly residueswere very flexible and ignored in the current analysis.A comparison of the two sets of data allows one todetermine which conformations were not observedduring the explicit solvent simulations. The 10 nsexplicit simulation sampled all of the above dihedralmaxima except for the maximum at 60° for �4 and �5,and the maximum centered at �60° for 5

2. However,these appear to be states with very low intrinsic pop-

ulations. Hence, the explicit solvent neutral pH sim-ulation sampled all the major local minima on thepeptide free energy surface. This suggests that most ofthe highly populated conformations should have beengenerated during the explicit solvent simulation atneutral pH, although the exact relative populationscould be subject to some error.

Comparison with NMR Data

Several NMR studies of Leu enkephalin in aqueoussolution have been performed. An early focus in-volved the degree of hydrogen bonding of the Glycarbonyl groups.15,17 Results obtained from 17O NMRindicate that the hydration of the carbonyl oxygens issimilar at all values of pH, and therefore they cannotbe involved in hydrogen bonding. Intramolecular hy-drogen bonding of the Gly carbonyl groups was verylow during the current simulations. This is illustratedby the carbonyl oxygen to water hydrogen radialdistribution functions (rdfs) displayed in Figure 5.

FIGURE 2 Time histories for selected distances from the simulations at low and high values ofpH. See Figure 1 for definitions. Both simulations started from the folded conformation obtainedafter 5 ns of the neutral pH simulation.

Conformational Analysis of Leu Enkephalin 181

The rdfs for both Gly carbonyl oxygens displayed thesame features and were independent of pH. Waterhydrogen first shell (�0.25 nm) coordination numbersfor the carbonyl oxygen of Gly2 were 1.2, 1.2, and 1.3for the neutral, low, and high pH simulations, respec-tively. The corresponding numbers for Gly3 were 1.4,1.4, and 1.2. Consequently, the present results appearto be in good agreement with the NMR data. How-ever, an exact quantitative comparison between thesimulation and NMR data is difficult to make.

The available NMR data also indicates that hydra-tion of the carboxylate terminus is relatively indepen-dent of changes between neutral and high pH.14 Todetermine the degree of hydration in the current sim-ulations, we have calculated the carboxylate oxygento water hydrogen rdfs (see Figure 5). Average firstshell (�0.27nm) hydrogen coordination numberswere 3.6, 0.8, and 4.1 for the neutral, low, and highpH simulations, respectively. The hydrogen coordina-tion numbers for the carboxylate oxygens in thefolded and unfolded states of the neutral pH simula-

tion were 2.8 and 4.3, respectively. The present resultsindicate that, while a sizable change in the hydrationof the oxygens between the folded and unfolded stateswas evident (2.8 vs 4.3), only small changes betweenthe neutral and high pH structures are predicted afteraveraging over the population of the folded and un-folded states (3.6 vs 4.1). Hence, while the currentneutral pH simulation was not long enough to deter-mine a precise population for the folded conforma-tion, it is clearly possible that the average degree ofhydration of the carboxylate oxygens can be relativelyindependent of pH even though an appreciable degreeof folding does occur at neutral pH.

Additional 14N NMR data indicates that the hydra-tion of the N-terminus is also unaffected on goingfrom neutral to low pH.27 The water oxygen first shell(�0.38 nm) coordination numbers were 5.0 and 5.1for the neutral and low pH simulations, respectively.Consequently, even though the folded form observedduring the current simulations adopted a close (0.48nm) head-to-tail arrangement; this had little effect on

FIGURE 3 Time histories for the major dihedral angles of Leu enkephalin at neutral pH.

182 Aburi and Smith

the hydration of the amino terminus. The agreementwith the experimental NMR data also extends to theLeu amide proton, which is known not to from hy-drogen bonds.16 Hydrogen bonds from the Leu amidegroup totaled less than 5% of the trajectory during theneutral pH simulation.

Comparison with Other Simulations

The most significant previous simulation of Leu en-kephalin at neutral pH in aqueous solution was per-formed by van der Spoel and Berendsen.5 They alsoobserved the formation of a folded structure thatstrongly resembles the one observed here. However,we also observed a significant interaction between theTyr ring and the Leu side chain, which may be a resultof the recent refinement of the hydrocarbon force fieldterms.28 In addition, both folded and unfolded con-formations occurred during the same simulation,whereas the previous study observed only the initial

conformations from which the simulation was started.Furthermore, they did not consider the effect of pH onthe conformations of Leu enkephalin. Hence, we con-sider the present results to be an improvement in ourcurrent understanding of the conformational charac-teristics of Leu enkephalin as described by the GRO-MOS force field.

Cluster Analysis

NMR studies of flexible peptides are complicated bythe number of possible conformations available tothese molecules in solution. In principle, moleculardynamics simulations can provide valuable informa-tion concerning the population distribution of thesepeptides. One way to classify these conformationsis by sorting into clusters of similar structures.Ideally, one would like to perform a cluster analysison trajectories which sample many reversible eventsto ensure good statistics and a correct ordering (by

FIGURE 4 Dihedral angle probability distributions for the major dihedrals of Leu enkephalin atneutral pH obtained from the explicit solvent molecular dynamics (MD) and implicit solventstochastic dynamics (SD) simulations.

Conformational Analysis of Leu Enkephalin 183

population) of the clusters.26 This is not the case forour simulation. However, the clustering analysis doesprovide a very convenient and simple way to describethe main conformational features of the peptide and itis used here for that purpose.

A cluster analysis of the neutral pH simulation wasperformed as described in the Methods section. Thenumber and size of the clusters as determined by threedifferent fitting methods are displayed in Table III.The clusters were determined by either fitting all theatoms, just the backbone atoms, or just the pharma-cophore groups (N-terminus and the two aromaticrings). The cluster number time histories for the threefitting methods are presented in Figure 6. The dihedralangles corresponding to the three major all atom clus-ters are presented in Table IV, with the most repre-sentative structures displayed in Figure 7. The threemajor all atom clusters corresponded to the folded(clusters 1 and 3) and unfolded (cluster 2) conforma-tions with changes in side chain dihedrals leading tothe population of other clusters. The two folded forms(1 and 3) differed in the orientation of 4

1 and a

rotation of the peptide group between the two Glyresidues. Cluster 2 involved the hydrophobic contactbetween the Tyr ring and the Leu side chain.

The backbone cluster number time history alsodisplayed two major clusters corresponding to the

FIGURE 5 Radial distribution functions for water hydrogens around the Gly carbonyl oxygensand the carboxylate oxygens, and for water oxygens around the N-terminal nitrogen as a functionof pH. All data correspond to the simulations starting from an unfolded conformation.

Table III Cluster Analysis for Leu Enkephalin atNeutral pHa

ClusterAll

Atoms Backbone Pharmacophore

1 15 49 432 13 31 123 4 6 104 3 5 75 3 3 4

Total numberof clusters 112 15 28

a The most populated conformations were determined from ananalysis of 1000 structures generated from each simulation accord-ing to three different fitting schemes. The data refer to percentagepopulations over the trajectory.

184 Aburi and Smith

folded and unfolded forms. The backbone conforma-tion changed occasionally during the first half of thetrajectory where the folded conformation dominated.During the second half of the trajectory, where theunfolded form(s) dominated, the backbone conforma-tion of the peptide remained surprisingly similar.Hence, although one would be tempted to consider thepeptide as unfolded and therefore rather random innature, the backbone conformation was actually rela-tively ordered. This agrees with the results of Daura etal., who also observed relatively few disordered statesof a small � peptide in comparison with the totalnumber of possible states.29

Clustering based on the pharmacophore groupsproduced interesting results. The three groups occu-

pied many arrangements over the course of the tra-jectory. However, most of these were short-lived,with only a few arrangements dominating. In partic-ular, the major cluster was present for almost half ofthe trajectory, and was populated when the peptidebackbone was in both the folded and extended con-formations. This implies that many backbone confor-mational transitions occurred while maintaining thesame spatial arrangement of the aromatic rings. Thisseems reasonable as the rings are bulky groups thatwould have to move a substantial distance through thesolvent if they were to maintain their side chain di-hedral angles.

In order to determine the extent to which the sameconformations were sampled in the neutral, low, and

FIGURE 6 Major cluster number time histories from the simulation at neutral pH. Data corre-sponds to results obtained after fitting of either all atoms (AA), the backbone atoms (BB), or thepharmacophore atoms (Pharm).

Conformational Analysis of Leu Enkephalin 185

FIGURE 7 Representative structures obtained from a cluster analysis using an all atom (AA) fit(top) and a backbone (BB) atom fit (bottom) of the neutral pH simulation. The top three clusters aredisplayed in each case. Clusters 1 and 3 of the all atom fit correspond to folded structures with a 23 5 hydrogen bond. Cluster 2 illustrates the strong Tyr-Leu side-chain interaction in the unfoldedconformation. Backbone clustering illustrates the major folded (1), in addition to the major (2) andminor (3) unfolded conformations, respectively.

Table IV Dihedral Angles for the Representative Structures of Leu Enkephalinat Neutral pHa

Fitting AA AA AA BB BB BB

Cluster 1 2 3 1 2 3

Tyr� 143 159 144 149 136 1141 �172 �171 �177

Gly� �173 �104 �113 �173 �96 �58� 102 116 �116 70 136 �131

Gly� 100 100 �74 115 96 �82� �89 �152 �105 �83 �167 154

Phe� �123 �99 �61 �130 �74 �93� �34 127 �31 �18 135 1171 73 �73 �174

Leu� �122 �110 �151 �166 �159 �1051 �76 �168 �772 �167 �57 �169

aObtained from a cluster analysis using all atoms (AA) or backbone (BB) fitting (see Table III).

186 Aburi and Smith

high pH simulations, the trajectories from all threesimulations were cross-clustered according to a fit oftheir corresponding backbone atoms. The results arepresented in Table V. The majority of the highlypopulated clusters were sampled under all three pHconditions, the main exception being the folded state

generated at neutral pH (cluster 3 in Table V) whichwas not sampled by the low pH simulation.

A clustering analysis was also performed for thestochastic dynamics simulation. None of the all-atomclusters possessed more than 4 members, indicating ahigh degree of sampling. If we consider this simulationto have sampled nearly all the possible excluded volumeconformations available to the peptide, then the numberof clusters observed from the explicit solvent and sto-chastic simulations provides another measure of the de-gree of sampling provided by the 10 ns explicit simula-tion. The total number of clusters obtained from theexplicit/stochastic simulations were 112/135, 15/26, and28/31 for fitting of the all-atom, backbone, and pharma-cophore groups, respectively. Again, we consider this tobe a very reasonable degree of sampling for this system.

Pharmacophore Analysis

The distribution of pharmacophore distances observedfor the three simulations is displayed in Figure 8. Themost populated cluster based on the fitting of thepharmacophore groups possessed average N—R1,N—R4, and R1—R4 distances of 0.51, 1.08, and 0.88

FIGURE 8 Probability distributions for the pharmacophore distance as a function of pH. Allsimulations started from an unfolded structure.

Table V Cross Clustering of the Neutral, Low, andHigh pH Simulationsa

Cluster Neutral pH Low pH High pH

1 3 10 572 29 38 23 48 0 14 1 13 115 1 14 16 4 5 57 4 7 18 1 2 49 0 0 6

10 1 4 2

a Percentage populations after backbone clustering of 1000structures from each of the simulations starting from an unfoldedconformation.

Conformational Analysis of Leu Enkephalin 187

nm, respectively, with an R1—N—R4 angle of 54°.These values coincide with the most probable dis-tances obtained from the neutral pH simulation (seeTable II and Figure 8). The distance distributionsvaried significantly between the different pH condi-tions, especially between the neutral and high pHsimulations. Most noticeable was the large probabilityfor close contact between the two aromatic rings athigh pH, which was negligible at neutral and low pH.

CONCLUSIONS

We have used molecular dynamics simulations toinvestigate the conformations accessible to Leu en-kephalin in aqueous solution at three different pHvalues. The simulations suggest that the peptide existsas a mixture of folded and unfolded forms of roughlyequal population at neutral pH. The folded form pos-sessed a 23 5 hydrogen bond with a close head to tailarrangement, and only displayed significant stabilityat neutral pH. Changes in hydration of the Gly car-bonyl oxygens and the terminal groups were deter-mined as a function of pH and found to be negligible,in agreement with the most recent NMR data.

The neutral pH simulation sampled many unfoldedstates of the peptide backbone. However, most ofthese were short-lived and one major form dominatedfor 30% of the 10 ns simulation. This illustrates that,even when peptides are assumed to have no recogniz-able structure, they are not necessarily totally randomand may adopt only a small fraction of the largenumber of possible conformations. One of the reasonsfor this was the presence of a strong hydrophobicinteraction between the Tyr and Leu side chains.

An analysis of the pharmacophore distributionsindicated that one arrangement dominated for 43% ofthe trajectory, and was observed for both the foldedand unfolded conformations of the peptide backbone.This suggests that the exact backbone conformation ofcyclic enkephalin derivatives does not necessarilyhinder the preferred relative orientation of the aro-matic rings, and therefore may not be a critical factorin activity of these molecules.

This project was supported by the Kansas AgriculturalExperimental Station (Publication 02-84-J).

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