Nmr and molecular modeling investigations of the neuropeptide bradykinin in three different solvent systems: DMSO, 9 : 1 dioxane/water,and in the presence of 7.4 mM lyso phosphatidylcholine micelles

The linear nonapeptide hormone bradykinin (Arg¹-Pro²-Pro³-Gly⁴-Phe⁵-Ser⁶-Pro⁷-Phe⁸-Arg⁹) is involved, either directly or indirectly, in a wide variety of physiological processes, particularly pain and hyperanalgesia. Additional evidence suggests that bradykinin also plays a major role in inflammatory response, asthma, sepsis, and symptoms associated with the rhinoviral infection. It has long been speculated that a β-turn at the C-terminus of bradykinin plays a major role in the biological activity of the neuropeptide. The β-turn forming potential of bradykinin in three vastly different local chemical environments, DMSO, 9 : 1 dioxane/water, and in the presence of 7.4 mM lyso phosphatidylcholine micelles, was investigated using two-dimensional homonuclear nmr experiments coupled with simulated annealing calculations. The results of these investigations show that in all three systems residues 6–9 of the C-terminus adopt very similar β-turn like structures. These results suggest that the β-turn at the C-terminus of bradykinin is an important secondary structural feature for receptor recognition and binding. © 1994 John Wiley & Sons, Inc.     

A proton magnetic resonance study of two synthetic agonist–antagonist pairs of bradykinin analogues

The conformation of two agonist–antagonist pairs of bradykinin (Arg¹-Pro²-Pro³-Gly⁴-Phe⁵-Ser⁶-Pro⁷-Phe⁸-Arg⁹) analogues were studied in CD₃OH/H₂O solution by ¹H-nmr techniques. The first agonist peptide studied, D -Arg⁰-Arg¹-Pro²-Hyp³-Gly⁴-Thi⁵-Ser⁶-Pro⁷-Thi⁸-Arg⁹, differs from the bradykinin sequence by the addition of D -Arg⁰, the replacement of the Phe moieties in positions 5 and 8 by Thi (Thi = β-(2-thienyl)-L -alanine), and Hyp³ (Hyp = L -4-hydroxy-L -proline) in position 3. In the corresponding antagonist sequence, Pro⁷ is replaced by D -Phe⁷. The second agonist–antagonist pair studied does not contain the D -Arg⁰ residue, which is present only to slow down the rate of metabolism. Based on complete resonance assignments from two-dimensional total correlation spectroscopy and rotating frame nuclear Overhauser effect spectroscopy spectra at 500 MHz, the peptides were analyzed in terms of intraresidue, sequential, and medium-range nuclear Overhauser effects, amide proton temperature coefficients, and vicinal coupling constants. Both agonist peptides show clear evidence for the existence of a type I β-turn comprising the C-terminal residues Ser⁶-Pro⁷-Thi⁸-Arg⁹ in fast conformational equilibrium with extended structures throughout. Although the conformational space is dominated by extended structures, the presence of the β-turn is spectroscopically clearly discernible. The two antagonist peptides, on the other hand, do not show evidence of turn formation but rather the presence of an extended conformation with some irregularities in the N-terminal region of the peptide. While the existence of a turn at the C-terminal end of bradykinin and its analogues with agonist activity has been predicted by empirical calculations and measurements in very apolar solvents, this study, for the first time, provides evidence based on physical data in a polar solvent environment that the turn is present, that it is type I and that it is essential for agonist activity. In the particular solvent used in these studies, the Pro⁷ to D -Phe⁷ substitution precluded the formation of the turn for the C-terminal residues of the antagonist. © 1993 John Wiley & Sons, Inc.     

Conformational Studies of Two Bradykinin Antagonists by Using Two-dimensional NMR Techniques and Molecular Dynamics Simulations

Abstract

The solution conformations of two potent antagonists of bradykinin (Arg¹-Pro²-Pro³-Gly⁴- Phe⁵-Ser⁶-Pro⁷-Phe⁸-Arg⁹), [Aca-¹, DArg⁰, Hyp³, Thi⁵, DPhe⁷,(N-Bzl)Gly⁸]BK (1) and [Aaa- ¹, DArg⁰, Hyp³, Thi⁵,(2-DNal)⁷, Thi⁸]BK (2), were studied by using 2D NMR spectroscopy in DMSO-dg and molecular dynamics simulations. The NMR spectra of peptide 1 reveals the existence of at least two isomers arising from isomerization across the DPhe⁷-(N-Bzl)Gly⁸peptide bond. The more populated isomer possesses the cis peptide bond at this position. The ratio of cis/trans isomers amounted to 7:3. With both antagonists, the NMR data indicate a β-turn structure for the Hyp³-Gly⁴ residues. In addition, for peptide 2, position 2,3 is likely to be occupied by turn-like structures. The cis peptide bond between DPhe⁷ and (N- Bzl)Gly⁸ in analogue 1 suggests type VI β-turn at position 7,8. The molecular dynamics runs were performed on both peptides in DMSO solution. The results indicate that the structure of peptide 1 is characterized by type VIb β-turn comprising residues Ser-Arg⁹ and the βI or βII-turn involving the Pro²-Thi⁵ fragment, whereas peptide 2 shows the tendency towards the formation of type I β-turn at position 2,3. The structures of both antagonists are stabilized by a salt bridge between the guanidine moiety of Arg¹ and the carboxyl group of Arg⁹. Moreover, the side chain of DArg⁰ is apart of the rest of molecule and is not involved in structural elements except for a few calculated structures.     

Truncation of the peptide sequence in bifunctional ligands with mu and delta opioid receptor agonist and neurokinin 1 receptor antagonist activities

The optimization and truncation of our lead peptide-derived ligand TY005 possessing eight amino-acid residues was performed. Among the synthesized derivatives, NP30 (Tyr¹-DAla²-Gly³-Phe⁴-Gly⁵-Trp⁶-O-[3′,5′-Bzl(CF₃)₂]) showed balanced and potent opioid agonist as well as substance P antagonist activities in isolated tissue-based assays, together with significant antinociceptive and antiallodynic activities in vivo.     

Conformational mimicry: Synthesis and solution conformation of a cyclic somatostatin hexapeptide containing a tetrazole cis amide bond surrogate

Potent, cyclic hexapeptide analogues of somatostatin are generally believed to adopt some common secondary structural features: a II′ β turn at one end of the cycle, and a type VI turn with a cis amide bond at the other. A proposed cis amide surrogate, the 1,5-disubstituted tetrazole, has been placed into a cyclic hexapeptide analog of somatostatin in order to constrain the putative cis amide bond. The final cyclization was done by either chemical or enzymatic means. The product, cyclo(Ala⁶-Tyr⁷-D -Trp⁸-Lys⁹-Val¹⁰-Phe¹¹-Ψ[CN₄]), was found to have 83% of the activity of somatostatin. Solution nmr analysis in DMSO/water revealed that the backbone as well as side chain χ₁ and χ₂ were well ordered. Relaxation matrix methods were used to extract distance restraints from the nuclear Overhauser effect spectroscopy data set, and these were used in a systematic search of torsional space to identify structures consistent with the nmr data. Restrained minimizations of these structures using a number of different force fields produced structures having the expected βII′ turn at D -Trp⁸-Lys⁹ and αβVIa turn in the Phe¹¹-Ψ[CN₄]-Ala⁶ portion of the molecule. The similarity of the minimized structures to those previously reported for cyclic hexapeptide analogues of somatostatin confirms the similarity of the tetrazole geometry to that of the cis amide in solution. © 1995 John Wiley & Sons, Inc.     

Conformational studies of human islet amyloid peptide using molecular dynamics and simulated annealing methods

Molecular dynamics simulations and simulated annealing in vacuum, model aqueous solution, and simulated membrane were used to analyze the conformational preferences of a segment spanning 20–29 residues of human islet amyloid polypeptide, [referred to as IAPP^H(20–29)]. Molecular dynamics simulations were conducted at 300 K on IAPP^H(20–29). The minimum energy conformers obtained in model aqueous solution and vacuum exhibited similar structures. Even in the absence of any constraints on peptide bonds, trans conformation was preferred consistently by all the peptide bonds. Analysis of the minimum energy conformers indicated that IAPP^H(20–29) showed a strong preference for turn structures in all the environments. These turn structures were stabilized by the formation of hydrogen bonds between the backbone amide and carbonyl groups. A good agreement was found between the results obtained from the molecular dynamics simulation and solid-state nmr experimental studies. © 1998 John Wiley & Sons, Inc. Biopoly 45: 9–20, 1998     

Proton magnetic resonance studies of bradykinin antagonists

Bradykinin (BK) is a peptide hormone with sequence Arg¹-Pro²-Pro³-Gly⁴-Phe⁵-Ser⁶-Pro⁷-Phe⁸-Arg⁹ and has been implicated in a multitude of pathophysiological processes such as the ability to lower systemic blood pressure and stimulate pain. BK analogues having bulky, β-branched D -aliphatic residues at position 7 combined with bulky L -aliphatic residues at position 8 have now been observed to be strong antagonists. Conformational studies based on two-dimensional nmr experiments in methanol/water (80/20 v/v) were carried out on several such active antagonists in a polar solvent. Included in this study were the very active antagonists, [D -Arg⁰, Hyp³, Thi⁵, D -Cpg⁷, Cpg⁸]-BK [Cpg: α-cyclo-pentyl-glycine; Hyp: trans-4-hydroxy-L -proline; Thi: β-(2-thienyl)-L -alanine] ( I ), [D -Arg⁰, Hyp³, D -Cpg⁷, Cpg⁸] -BK ( II ), as well as its variant with D -Cpg⁷ replaced by Cpg⁷, namely [D -Arg⁰, Hyp³, Cpg⁷, Cpg⁸]-BK ( III ). A turn-like structure, which coexists with the extended conformation, was observed between residues 2 and 5 for the most active antagonists I and II , in direct correlation with the peptide activities. No turn-like structure was found for residues 6–9. In peptide III , a turn-like structure was not identified. The existence of a turn at the C-terminal end of bradykinin and its analogues has been predicted by empirical calculations and supported by nmr measurements. But the present nmr study on the most active antagonists ( I , II ) does not support this hypothesis. Instead, the data suggest that a turn-like structure between residues 2 and 5 could be important for antagonist activity. Finally, one weak inhibitor [D -Cpg⁷]-BK ( IV ) showed no defined secondary structure. © 1993 John Wiley & Sons, Inc.     

Studies of conformational isomerism in α-melanocyte stimulating hormone by design of cyclic analogues

Results of energy calculations for α-MSH (α-melanocyte stimulating hormone, Ac-Ser¹-Tyr²-Ser³-Met⁴-Glu⁵-His⁶-Phe⁷-Arg⁸-Trp⁹-Gly¹⁰-Lys¹¹-Pro¹²-Val¹³-NH₂) and [D -Phe⁷]α-MSH were used for design of cyclic peptides with the general aim to stabilize different conformational isomers of the parent compound. The minimal structural modifications of the conformationally flexible Gly¹⁰ residue, as substitutions for L -Ala, D -Ala, or Aib (replacing of hydrogen atoms by methyl groups), were applied to obtain octa- and heptapeptide analogues of α-MSH(4–11) and α-MSH(5–11), which were cyclized by lactam bridges between the side chains in positions 5 and 11. Some of these analogues, namely those with substitutions of the Gly¹⁰ residue with L -Ala or Aib, showed biological activity potencies on frog skin comparable to the potency of the parent tridecapeptide hormone. Additional energy calculations for designed cyclic analogues were used for further refinement of the model for the biologically active conformations of the His-Phe-Arg-Trp “message” sequence within the sequences of α-MSH and [D -Phe⁷]α-MSH. In such conformations the aromatic moieties of the side chains of the His⁶, L/D -Phe⁷, and Trp⁹ residues form a continuous hydrophobic “surface,” presumably interacting with a complementary receptor site. This feature is characteristic for low-energy conformers of active cyclic analogues, but it is absent in the case of inactive analogues. This particular spatial arrangement of functional groups involved in the message sequence is very close for α-MSH and [D -Phe⁷]α-MSH, as well as for biologically active cyclic analogues despite differences of dihedral angle values for corresponding low-energy conformations. © 1998 John Wiley & Sons, Inc. Biopoly 46: 155–167, 1998     

Existence of Met-enkephalin-Arg6-Gly7-Leu8 with Met-enkephalin,Leu-enkephalin and Met-enkephalin-Arg6-Phe7 in the brain of guinea pig,rat and golden hamster

High performance liquid chromatography (HPLC) coupled with specific radioimmunoassays for methionine-enkephalin-Arg⁶-Gly⁷-Leu⁸ (Met-E-Arg⁶-Gly⁷-Leu⁸), methionine-enkephalin (Met-E), leucine-enkephalin (Leu-E) and methionine-enkephalin-Arg⁶-Phe⁷ (Met-E-Arg⁶-Phe⁷) has demonstrated that Met-E-Arg⁶-Gly⁷-Leu⁸ exists together with Met-E, Leu-E and Met-E-Arg⁶-Phe⁷ in the brain of guinea pig, rat and golden hamster. The content of Met-E-Arg⁶-Gly⁷-Leu⁸ was comparable to those of Leu-E and Met-E-Arg⁶-Phe⁷, whereas that of Met-E was the highest among the four opioid peptides. These results are compatible with the recent studies on the nucleotide sequence of cloned cDNA for preproenkephalin from bovine adrenal medulla, which reveal that this precursor molecule contains four copies of Met-E and one copy each of Leu-E, Met-E-Arg⁶-Phe⁷ and Met-E-Arg⁶-Gly⁷-Leu⁸. The co-existence of Met-E-Arg⁶-Gly⁷-Leu⁸ with Met-E, Leu-E and Met-E-Arg⁶-Phe⁷ suggests that their biosynthetic pathway in the brain is similar to that in the adrenal medulla.     

Nmr and molecular modeling investigations of the neuropeptide substance P in the presence of 15 mM sodium dodecyl sulfate micelles

To better understand the structural basis of the biological activity of the neuropeptide substance P SP; (Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-NH₂), two-dimensional nmr spectroscopy experiments and simulated annealing calculations were used to investigate the conformation adopted in the presence of the membrane model system sodium dodecyl sulfate. It was determined that SP in the presence of SDS micelles undergoes a conformational equilibrium between an α- and a 3₁₀-helix involving the midregion (Pro⁴-Gln⁵-Gln⁶-Phe⁷-Phe⁸) of the peptide. The C-terminus adopts an extended conformation while the N-terminus remains quite flexible. The conformation adopted by SP in the presence of SDS micelles yields a structure that is consistent with the model of a neurokinin-1 selective ligand proposed by Convert. © 1994 John Wiley & Sons, Inc.     

Preferred solution and calculated conformations of dermorphin and analysis of structure–conformation–activity relationships in the series [A1an]-dermorphin

We describe the solution (¹H-nmr) and calculated conformations of the opiatelike peptide dermorphin and the analysis of structure–conformation–activity relationships in the series [Alaⁿ]-dermorphin. We used ¹H-nmr spectroscopy to study dermorphin and its analogs [Alaⁿ]-dermorphin (with n = 1, 2…7) dissolved in dimethylsufoxide. Conformational energy calculations using semiempirical partitioned energy function methods were then carried out on dermorphin and its [L -Ala²]-analog. Agreement between calculation and experiment is satisfying, both suggesting predominance of a type I β-turn around Pro⁶-Ser⁷ at the C-terminus and of an extended structure in the central sequence Phe³-Gly⁴-Tyr⁵. Detailed analysis by step-by-step substitutions with Ala indicates that intraresidue interactions dominate over medium-range interactions (between adjacent residues), although the latter may also have a noticeable influence in shaping conformations. As a general feature, the effects of substitutions on the arrangement of side chains are always larger on the succeeding residue than on the preceding residue. Almost all the variations of activity observed in the analogs can be explained from conformational changes occurring in the aromatic side chains of the biologically important Tyr¹, Phe³, and Tyr⁵ on substitutions effected on adjacent residues (fluctuations via medium-range interactions).     

Occurrence of met-enkephalin,met-enkephalin-Arg6-Phe7 and met-enkephalin-Arg6-Gly7-Leu8 in gastrin cells of hog antral mucosa

Summary Region-specific antisera to three enkephalins: met-enkephalin, met-enkephalin-Arg⁶-Phe⁷ and met-enkephalin-Arg⁶-Gly⁷-Leu⁸, together with four region specific antisera to progastrin: C-terminal G17 specific, N-terminal G34 specific, cryptic peptides A- and B-specific, were used in immunohistochemical studies of hog antral mucosa. A sub-population (6–10%) of the gastrin-containing endocrine cells (G-cells) was found to react with antisera to met-enkephalin, met-enkephalin-Arg⁶-Phe⁷ and met-enkephalin-Arg⁶-Gly⁷-Leu⁸. About 30% of all the enkephalin-containing cells were identified as G-cells. The results indicate that a fraction of G-cells produces both enkephalin-like peptides and gastrin.     

Crystal structures of two cyclic pseudopentapeptides containing ψ[CH2S] and ψ[CH2SO] backbone surrogates

The solid state conformations of cyclo[Gly–Proψ[CH₂S]Gly–D –Phe–Pro] and cyclo[Gly–Proψ[CH₂–(S)–SO]Gly–D –Phe–Pro] have been characterized by X-ray diffraction analysis. Crystals of the sulfide trihydrate are orthorhombic, P2₁2₁2₁, with a = 10.156(3) Å, b = 11.704(3) Å, c = 21.913(4) Å, and Z = 4. Crystals of the sulfoxide are monoclinic, P2₁, with a = 10.662(1) Å, b = 8.552(3) Å, c = 12.947(2) Å, β = 94.28(2), and Z = 2. Unlike their all-amide parent, which adopts an all-trans backbone conformation and a type II β-turn encompassing Gly-Pro-Gly-D -Phe, both of these peptides contain a cis Gly¹-Pro² bond and form a novel turn structure, i.e., a type II′ β-turn consisting of Gly–D –Phe–Pro–Gly. The turn structure in each of these peptides is stabilized by an intramolecular H bond between the carbonyl oxygen of Gly¹ and the amide proton of D -Phe⁴. In the cyclic sulfoxide, the sulfinyl group is not involved in H bonding despite its strong potential as a hydrogen-bond acceptor. The crystal structure made it possible to establish the absolute configuration of the sulfinyl group in this peptide. The two crystal structures also helped identify a type II′ β-turn in the DMSO-d₆ solution conformers of these peptides. © 1993 John Wiley & Sons, Inc.     

Solution conformational preferences of a peptidic analogue of a natural macrolide

The conformational behavior in solution of a cyclic peptide with sequence cyclo-(Pro¹-Pro²-Dab³ (cHexA)ψ[N⁷HCO]-Leu⁴ψ[NHCO]-Suc⁵-Gly⁶-) has been throughly investigated with the combined use of nmr and molecular dynamic techniques. The compound, which has been modeled to mimic the FK506 macrolide bound to the FK506 binding protein structure, can be considered as a peptidic analogue of the FK506 system. The synthesis was carried out on a phenylacetoamidomethyl resin using an appropriate protocol for the peptide chain elongation. The conformational properties of the cyclic hexapeptide were studied in DMSO and water. The nmr data in DMSO and restrained molecular dynamics simulations show the presence of two contiguous cis peptide bonds involving the -Gly-Pro-Pro- segment. This segment in water exhibits conformational heterogeneity presenting at least two distinct conformational families, characterized the first by cis-cis and the second by a trans-cis Gly-Pro-Pro configuration; the trans-cis isomer was fully characterized. © 1997 John Wiley & Sons, Inc. Biopoly 42: 349–361, 1997     

β-Alanine containing cyclic peptides with predetermined turned structure. V

In the present paper we describe the synthesis, purification, single crystal x-ray analysis, and solution structural characterization by nmr spectroscopy, combined with restrained molecular dynamic simulations, of the cyclic hexapeptide cyclo-(Pro-Phe-β-Ala-Phe-Phe-β-Ala). The peptide was synthesized by classical solution methods and the cyclization of the free hexapeptide was accomplished in good yields in diluted methylenechloride solution using N, N-dicyclohexyl-carbodiimide. The compound crystallizes in the monoclinic space group P2₁ from methanol/ethyl acetate. The molecule adopts in the solid state a conformation characterized by cis β-Ala⁶-Pro¹ peptide bond. The α-amino acid residues are at the corner positions of turned structures. The Pro¹-Phe² segment is incorporated in a pseudo type I β-turn, while Phe⁴-Phe⁵ is in a typical type I β-turn. Assignment of all ¹H and ¹³C resonances was achieved by homo- and heteronuclear two-dimensional techniques in dimethylsulfoxide (DMSO) solutions. The conformational analysis was based on inter-proton distances derived from rotating frame nuclear Overhauser effect spectroscopy spectra and homonuclear coupling constants. Restrained molecular dynamic simulation in vacuo was also performed to built refined molecular models. The molecule is present in DMSO solution as two slowly interconverting conformers, characterized by a cis-tran isomerism around the β-Ala⁶-Pro¹ peptide bond. This work confirms our expectations on the low propensity of β-alanyl residues to be positioned at the corners of turned structure. © 1994 John Wiley & Sons, Inc.     

Multiple interconverting conformers of the cyclic tetrapeptide tentoxin, [cyclo-(L-MeAla1-L-Leu2-MePhe[(Z)Δ]3-Gly4)], as seen by two-dimensional 1H-nmr spectroscopy

The conformations of the phytotoxic cyclic tetrapeptide tentoxin [cyclo-(L -MeAla¹-L -Leu²-MePhe[(Z)Δ]³-Gly⁴ )] have been studied in aqueous solution by two-dimensional proton nmr at various temperatures. Contrary to what is observed in chloroform, tentoxin exhibits multiple exchanging conformations in water. Aggregation phenomena were also observed. Four conformations with different proportions (51, 37, 8, and 4%) were observed at ?5°C. Models were constructed from nmr parameters and restrained molecular dynamics simulations. All the models exhibit cis-trans-cis-trans conformation of the amide bond sequence. The conversion from one form to another is accomplished by a conformational peptide flip consisting of a 180° rotation of a nonmethylated peptide bond. © 1995 John Wiley & Sons, Inc.     

The structure of α-amanitin in dimethylsulfoxide solution

The backbone and side-chain conformations of the bicyclic octapeptide α-amanitin indimethylsulfoxide (DMSO) solution have ben deduced from analysis of the nmr spectrl parameters and conformational energy calculations. Several ambiguities in the nmr spectral assignments were resolved following a comparison with the recently published conformation of β-amanitin in the crystalline state. The peptide proton exchange and temperature coefficient data demonstrate strong intramolecular hyfrogen bonds for the GLY⁵ and Cys⁸ peptide protons. The vicinal proton coupling constants are consistent with the cyclic octapeptide udergoing chain reversl at the Ile⁶-Gly⁷ abd the Hyp²-Hyi³ dipeptide segments. The upfield shifts of the glycine and isoleucine protons demonstrate the folding of the indole ring of the Trp⁴-Cys⁸ brifge towards the Gly⁵-Ile⁶-Gly⁷ half of the Ile-amanitin molecule. The structure af α-amanitin in DMSO is defined by the (?ψ) backbone rotation angles Trp⁴(?90, ?60), Gly⁵ (+120, ?120), Ile⁶(?6, +120), Gly⁷ (+45, +60), Cys⁸(?120, ?60), Asn¹ (+175, ?175), Hyp² (?160, ?45), and Hyi³ (?90, ?60). The study demonstrates that the structure of α-amanitin in solution is similar to the structure f β-amanitin in the crystalline state.     

Prediction of the native conformation of a polypeptide by a statistical-mechanical procedure. III. Probable and average conformations of enkephalin

The program SMAPPS (Statistical-Mechanical Algorithm for Predicting Protein Structure) was originally designed to determine the probable and average backbone (?, ψ) conformations of a polypeptide by the application of equilibrium statistical mechanics in conjunction with an adaptive importance sampling Monte Carlo procedure. In the present paper, the algorithm has been extended to include the variation of all side-chain (χ) and peptide-bond (ω) dihedral angles of a polypeptide during the Monte Carlo search of the conformational space. To test the effectiveness of the generalized algorithm, SMAPPS was used to calculate the probable and average conformations of Met-enkephalin for which all dihedral angles of the pentapeptide were allowed to vary. The total conformational energy for each randomly generated structure of Met-enkephalin was obtained by summing over the interaction energies of all pairs of nonbonded atoms of the whole molecule. The interaction energies were computed by the program ECEPP /2 (Empirical Conformational Energy Program for Peptides). Solvent effects were not included in the computation. The results of the Monte Carlo calculation of the structure of Met-enkephalin indicate that the thermodynamically preferred conformation of the pentapeptide contains a γ-turn involving the three residues Gly²-Gly³-Phe⁴. The γ-turn conformation, however, does not correspond to the structure of lowest conformational energy. Rather, the global minimum-energy conformation, recently determined by a new optimization technique developed in this laboratory, contains a type II′ β-bend that is formed by the interaction of the four residues Gly²-Gly³-Phe⁴-Met⁵. A similar minimum-energy conformation is found by the SMAPPS procedure. The thermodynamically preferred γ-turn structure has a conformational energy of 4.93 kcal/mole higher than the β-bend structure of lowest energy but, because of the inclusion of entropy in the SMAPPS procedure, it is estimated to be ～ 9 kcal/mole lower in free energy. The calculation of the average conformation of Met-enkephalin was repeated until a total of ten independent average conformations were established. As far as the phenylalanine residue of the pentapeptide is concerned, the results of the ten independent average conformations were all found to lie in the region of conformational space corresponding to the γ-turn. These results further support the conclusion that the γturn conformation is thermodynamically favored.     

D-isomeric replacements within the 6-9 core sequence of Ac-[Nle4]-alpha-MSH4-11-NH2: a topological model for the solution conformation of alpha-melanotropin

Analogs of Ac-[Nle⁴]-α-MSH_4–11-NH₂ and Ac-[Nle⁴, D -Phe⁷]-α-MSH_4–11-NH₂ were prepared with D -isomeric replacements at the His⁶, Arg⁸, and Trp⁹ residues. The requirement for an indole moiety at position 9 also was evaluated by replacement with L -leucine in both parent fragment analogs. D -isomeric replacements at positions 6 and 8 in either series were detrimental to biological potency in frog (Rana pipiens) and lizard skin (Anolis carolinensis) in vitro melanotropic assays. However, Ac-[Nle⁴, D -Trp⁹]-α-MSH_4–11-NH₂ and Ac-[Nle⁴, D -Phe⁷, D -Trp⁹]-α-MSH_4–11-NH₂ were equipotent and 10 × more potent than Ac-[Nle⁴]-α-MSH_4–11-NH₂, respectively, in the lizard skin bioassay, and 30 and 1900 times more potent in the frog skin bioassay. Ac-[Nle⁴, D -Phe⁷, D -Trp⁹]-α-MSH_4–11-NH₂ was 3 × more potent than α-MSH in the frog skin bioassay. Proton nmr studies in aqueous solution revealed a marked preservation of the backbone conformation of these linear analogs. Chemical-shift variations due to the through-space anisotropic influence of the core aromatic amino acid residues permitted evaluation of side-chain topology. The observed topology was consistent with nonhydrogen-bonded β-like structure (? = ?139°, ψ = +135° for L -amino acids; ? = +139°, ψ = ?135° for D -amino acids) as the predominant solution conformation. The biological and conformational data suggest that high melanotropic potency requires a close spatial arrangement of the His⁶, Phe⁷, and Arg⁸ side chains.     

Substrate specificity of thermitase,a thermostable serine proteinase fromThermoactinomyces vulgaris

The specificity of thermitase (EC 3.4.21.14), a microbial thermostable serine proteinase fromThermoactinomyces vulgaris, with several oligo- and polypeptide substrates was investigated. Preferred hydrolysis of peptide bonds with a hydrophobic amino acid at the carboxylic site was observed. The proved carboxypeptidolytic splitting of Leu⁵-enkephalin and bradykinin, as well as the noncleavability of casomorphins by thermitase, can be explained by the position of the glycine and proline residues in these substrates. Major cleavage sites in the oxidized insulin B chain in a 15-min incubation with thermitase at Gln⁴-His⁵, Ser⁹-His¹⁰, Leu¹¹-Val¹², Leu¹⁵-Tyr¹⁶ and in the oxidized insulin A chain at Cys SO₃H¹¹-Ser¹², Leu¹³-Tyr¹⁴, and Leu¹⁶-Glu¹⁷ were observed. Additional cleavages of the bonds His⁵-Leu⁶, Arg²²-Gly²³, Phe²⁴-Phe²⁵, Phe²⁵-Tyr²⁶, and Tyr²⁶-Thr²⁷ in the oxidized B chain and Cys SO₃H⁶-Cys SO₃H⁷ and Tyr¹⁹-Cys SO₃H²⁰ in the oxidized A chain in 2-h incubations with thermitase were also noted. Hydrolysis of salmine A I component in a 10-min incubation was observed mainly at four peptide bonds: Arg⁵-Ser⁶, Ser⁶-Ser⁷, Arg¹⁸-Val¹⁹, and Gly²⁷-Gly²⁸. The cleavage sites of thermitase in both insulin chains were similar to those reported in the studies of subtilisins.