The influence of steric interactions on the conformation and biology of oxytocin. Synthesis and analysis of penicillamine(6)-oxytocin and penicillamine(6)-5-tert-butylproline(7)-oxytocin analogs.

Six [Pen(6)]oxytocin analogs were synthesized by substituting penicillamine for cysteine in oxytocin, [Mpa(1)]oxytocin, [dPen(1)]oxytocin, [5-t-BuPro(7)]oxytocin, [Mpa(1), 5-t-BuPro(7)]oxytocin and [dPen(1), 5-t-BuPro(7)]oxytocin. When tested in the uterotonic test in vitro [Pen(6)]oxytocin, [Pen(6), 5-t-BuPro(7)]oxytocin, [Mpa(1), Pen(6)]oxytocin and [Mpa(1), Pen(6), 5-t-BuPro(7)]oxytocin, all were found to possess both agonistic and antagonistic properties. Their agonistic potency ranged from negligible (0.08 IU/mg) to low (5.85 IU/mg) and their antagonistic potency (pA2) was estimated to range from 6.6 to 7.9. [dPen(1), Pen(6)]Oxytocin and [dPen(1), Pen(6), 5-t-BuPro(7)]oxytocin were found to be pure antagonists with similarly high pA2 values of approximately 8.2. Replacement of proline by 5-tert-butylproline increased binding affinity by a factor of two in [Pen(6)]oxytocin and had no influence on the binding affinity of [Mpa(1), Pen(6)]oxytocin and [dPen(1), Pen(6)]oxytocin. Assignment of the proton signals for prolyl amide cis- and trans-isomers by NMR experiments in water indicated that the Pen(6)-5-tert-BuPro(7) peptide bond cis-isomer population was augmented relative to the prolyl peptides and measured, respectively, at 20, 35 and 35% in the 5-tert-butylproline(7) analogs of [Pen(6)]oxytocin, [Mpa(1), Pen(6)]oxytocin and [dPen(1), Pen(6)]oxytocin. This augmentation in cis-isomer population was correlated with a 21-fold reduction in the agonistic potency and 2-fold augmentation in antagonistic potency for [Pen(6), 5-t-BuPro(7)]oxytocin relative to [Pen(6)]oxytocin. Augmentation of cis-isomer population was also correlated to reduced agonist potency without effect on antagonism on conversion of [Mpa(1), Pen(6)]oxytocin to [Mpa(1), Pen(6), 5-t-BuPro(7)]oxytocin. In the potent oxytocin antagonist, [dPen(1), Pen(6)]oxytocin, substitution of 5-tert-butylproline for proline augmented the cis-isomer population without affecting antagonistic potency. The synthesis and evaluation of [Pen(6)]oxytocin and [Pen(6), 5-t-BuPro(7)]oxytocin analogs 1-6 indicated that steric interactions influenced agonist and antagonist activity by modifying peptide conformation. Augmentations in the prolyl cis-isomer population caused by 5-tert-butylproline occurred concurrently with enhanced or maintained antagonistic potency and binding affinity and reduced agonistic potency.     

Structure and conformation of the disulfide bond in dimeric lung surfactant peptides SP-B1-25 and SP-B8-25

Raman spectroscopy was used to determine the conformation of the disulfide linkage between cysteine residues in the homodimeric construct of the N-terminal alpha helical domain of surfactant protein B (dSP-B(1-25)). The conformation of the disulfide bond between cysteine residues in position 8 of the homodimer of dSP-B(1-25) was compared with that of a truncated homodimer (dSP-B(8-25)) of the peptide having a disulfide linkage at the same position in the alpha helix. Temperature-dependent Raman spectra of the S-S stretching region centered at approximately 500 cm(-1) indicated a stable, although highly strained disulfide conformation with a chi(CS-SC) dihedral angle of +/-10 degrees for the dSP-B(1-25) dimer. In contrast, the truncated dimer dSP-B(8-25) exhibited a series of disulfide conformations with the chi(CS-SC) dihedral angle taking on values of either +/-30 degrees or 85+/-20 degrees . For conformations with chi(CS-SC) close to the +/-90 degrees value, the Raman spectra of the 8-25 truncated dimers exhibited chi(SS-CC) dihedral angles of 90/180 degrees and 20-30 degrees . In the presence of a lipid mixture, both constructs showed a nu(S-S) band at approximately 488 cm(-1), corresponding to a chi(CS-SC) dihedral angle of +/-10 degrees . Polarized infrared spectroscopy was also used to determine the orientation of the helix and beta-sheet portion of both synthetic peptides. These calculations indicated that the helix was oriented primarily in the plane of the surface, at an angle of approximately 60-70 degrees to the surface normal, while the beta structure had approximately 40 degrees tilt. This orientation direction did not change in the presence of a lipid mixture or with temperature. These observations suggest that: (i) the conformational flexibility of the disulfide linkage is dependent on the amino acid residues that flank the cysteine disulfide bond, and (ii) in both constructs, the presence of a lipid matrix locks the disulfide bond into a preferred conformation.     

Desaminopenicillamine tocinoic acid derivatives--inhibitors of oxytocin

Tocinoic acid analogs with penicillamine in place of one or both of the cysteine residues have been studied and [1-beta-mercaptopropionic acid, 6-penicillamine] tocinoic acid (dPen6TA) and [1-beta,beta-dimethyl-beta-mercaptopropionic acid, 6-penicillamine] tocinoic acid (dPen1Pen6TA) have been synthesized in solution. Biological activities of these 2 compounds and those of the previously synthesized [1-beta,beta-dimethyl-beta-mercaptopropionic acid] tocinoic acid (dPen1TA) have been assayed. It was found that dPen1TA and dPen1Pen6TA, both of which have a beta,beta-dimethyl-beta-mercaptopropionic acid in position 1, are strong inhibitors of the uterine activity of oxytocin in vitro (without Mg2+) with pA2 values of 7.1 and 7.8, respectively, whereas dPen6TA with penicillamine in position 6 is a mild agonist.     

Penile erection and yawning induced by oxytocin and related peptides: structure-activity relationship

The potency of several oxytocin-related peptides in inducing penile erection and yawning after injection into a lateral ventricle of male rats was compared. Substitution of two amino acids in the oxytocin molecule or deletion of the C-terminal glycinamide as in des-GlyNH2-oxytocin [oxytocin(1-8)] reduced oxytocin potency in inducing both effects, the rank order being: oxytocin greater than [Thr4,Gly7]-oxytocin congruent to isotocin [( Ser4,Ile8]-oxytocin) greater than vasopressin [( Phe3,Arg8]-oxytocin) greater than des-GlyNH2-oxytocin. Oxytocin's ability to induce penile erection and yawning was abolished by permanent opening of the disulfide bridge by reduction and carboxymethylation. Oxytocin(1-6) and oxytocin(7-9) were also inactive. Penile erection and yawning induced by oxytocin-related peptides were antagonized in a dose-dependent manner by nonapeptide antagonists with a rank order of potency that follows their antioxytocic activity (d[(CH2)5Tyr(Me)Orn8]-vasotocin congruent to [Pen1,Phe(Me)2,Thr4,Orn8]-oxytocin greater than d[(CH2)5Tyr(Me)Arg8]-vasopression). Carboxymethylated oxytocin, oxytocin(1-6), and oxytocin(7-9) were devoid of antagonistic activity. The present results suggest that central oxytocin receptors mediating the expression of penile erection and yawning are structurally related to those present in the uterus and in the mammary gland.     

Conformational studies of [Abu(3, 11)]-SFTI-1, an analogue of the trypsin inhibitor isolated from sunflower seeds.

With only 14 amino acid residues, the trypsin inhibitor SFTI-1 is the smallest naturally occurring serine proteinase inhibitor. It consists of two cyclic fragments (with head-to-tail cyclization and a disulfide bridge). In our previous paper, we showed that the removal of the disulfide bridge produced 2.4-fold lower activity. Here, we present the total conformational analysis of the [Abu(3, 11)]-SFTI-1 analog by means of 2D NMR spectroscopy in conjunction with theoretical methods. The peptide was synthesized by Fmoc SPPS. It was cyclized with PyBop and DIPEA in DMF. The NMR studies were performed in DMSO-d(6) at 303 K. Conformations of the peptide studied were calculated by the following three approaches: distance geometry (DG), molecular dynamics (MD) and determination of the statistical weights of conformations. The first two algorithms use a CHARMM force field, whereas the last uses an ECEPP/3 force field. Our calculations resulted in three sets of conformers with 7, 9 and 6 representatives, respectively. All our results were compared with published ones. It was found that the peptide has an ill-defined structure. Despite its conformational flexibility, the binding loop (3-11 fragment) displayed geometry similar to the corresponding fragments of the other SFTI-1 analogs and to the inhibitor itself. Furthermore, the peptide bond between the Ile7 and Pro8 residues adopts cis geometry, which is essential for inhibitory activity. Copyright (c) 2008 European Peptide Society and John Wiley & Sons, Ltd.     

Structure and conformation of the disulfide bond in dimeric lung surfactant peptides SP-B1-25 and SP-B8-25

Raman spectroscopy was used to determine the conformation of the disulfide linkage between cysteine residues in the homodimeric construct of the N-terminal alpha helical domain of surfactant protein B (dSP-B_1-25). The conformation of the disulfide bond between cysteine residues in position 8 of the homodimer of dSP-B_1-25 was compared with that of a truncated homodimer (dSP-B_8-25) of the peptide having a disulfide linkage at the same position in the alpha helix. Temperature-dependent Raman spectra of the S-S stretching region centered at ∼ 500 cm^− 1 indicated a stable, although highly strained disulfide conformation with a χ(CS-SC) dihedral angle of ± 10° for the dSP-B_1-25 dimer. In contrast, the truncated dimer dSP-B_8-25 exhibited a series of disulfide conformations with the χ(CS-SC) dihedral angle taking on values of either ± 30° or 85± 20°. For conformations with χ(CS-SC) close to the ± 90° value, the Raman spectra of the 8-25 truncated dimers exhibited χ(SS-CC) dihedral angles of 90/180° and 20-30°. In the presence of a lipid mixture, both constructs showed a ν(S-S) band at ∼ 488 cm^− 1, corresponding to a χ(CS-SC) dihedral angle of ± 10°. Polarized infrared spectroscopy was also used to determine the orientation of the helix and β-sheet portion of both synthetic peptides. These calculations indicated that the helix was oriented primarily in the plane of the surface, at an angle of ∼ 60-70° to the surface normal, while the β structure had ∼ 40° tilt. This orientation direction did not change in the presence of a lipid mixture or with temperature. These observations suggest that: (i) the conformational flexibility of the disulfide linkage is dependent on the amino acid residues that flank the cysteine disulfide bond, and (ii) in both constructs, the presence of a lipid matrix locks the disulfide bond into a preferred conformation.     

Conformational analysis of [Cpp1, Sar7, Arg8] vasopressin by 1H-NMR spectroscopy and molecular mechanics calculations.

A combined 1H-NMR and molecular mechanics study of [Cpp1, Sar7]AVP was performed in order to select the most probable conformations in DMSO solutions. The NMR constraints obtained were employed in the selection of starting conformations of the cyclic moiety of the analog. In particular, the diminished accessibility of the Asn5 NH proton to solvent and the close contact between Cpp1 and Cys6 C alpha H protons suggests a beta-turn conformation at the Phe3-Gln4 residues. Energy minimization was carried out both in the ECEPP/2 (rigid-valence geometry) and in the AMBER (flexible-valence geometry) force fields. Comparison of the experimental and calculated values of NMR characteristics has revealed that conformations containing type I, II, and III beta-turns at the Phe3-Gln4 residues are in reasonable agreement with the experimental data, with a dynamic equilibrium between the beta I (beta III) and beta II type structures of the cyclic part being the most probable. All of these conformations prefer the negative chirality of the disulfide bridge (theta 3 approximately -90 degrees). Five representative conformations were chosen for the acyclic tail: one with a beta I, one with a beta II'-turn at the Sar7-Arg8 residues, two extended-type conformations, and a conformation with a gamma-turn at Sar7. Because only high-energy extended conformations were in agreement with NMR data, it was concluded that the acyclic tail has considerable conformational flexibility in solution. The conformations obtained are discussed in terms of the structure-function relationship of the neurohypophyseal hormone analogs.     

Conformational studies of somatostatin and selected analogues by Raman spectroscopy

Laser Raman spectroscopy has been used to study the conformations of somatostatin and some selected analogues in aqueous solution. The results indicate that the CS-SC dihedral angles of somatostatin and of these analogues (except [Ala3,14]-SS, which has no disulfide bond) are within 20 degrees of +/- 85 degrees, and the SS-CC dihedral angles are predominantly in the range of 50 degrees-180 degrees. Furthermore, from the behavior of the amide I' and amide III bands, it appears that somatostatin adopts a beta-pleated sheet structure, whereas its analogues are less ordered (to varying degrees).     

Conformation of D-Phe-Cys-Tyr-D-Trp-Lys-Thr-Pen-Thr-NH2 (CTP-NH2), a highly selective mu-opioid antagonist peptide, by 1H and 13C n.m.r

The 1H and 13C n.m.r. spectral parameters of CTP-NH2 [D-Phe-Cys-Tyr-D-Trp-Lys-Thr-Pen-Thr-NH2], a potent, highly selective mu-opiate antagonist, were measured in aqueous solution and a possible conformation has been deduced from the spectral data. The data are consistent with a type II' beta-turn for the tetrapeptide sequence -Tyr3-D-Trp4-Lys5-Thr6-. Solvent shielding of the Cys2 amide proton, observed in variable temperature experiments, suggests an orientation of this amide proton toward the gem dimethyls of Pen7 with possible hydrogen bonding to the Thr6 carbonyl oxygen, and a dihedral angle of -110 degrees for the disulfide bond. Partially relaxed Fourier transform 13C relaxation studies confirm a constrained cyclic system, with the C alpha carbons in the "hinge" of the beta-turn having the shortest t1 times. Segmental motion was observed for the side chain of Lys5.     

Conformational analyses of sandostatin analogs containing stereochemical changes in positions 6 or 8

We report the conformational analysis by 1H nmr in DMSO and computer simulations involving distance geometry and molecular dynamics simulations of analogs of the cyclic octapeptide D-Phe1-c[Cys2-Phe3-D-Trp4-Lys5-Thr6-Cys 7]-Thr8-ol (sandostatin, octreotide). The analogs D-Phe1-c[Cys2-Phe3-D-Trp4-Lys5-Xaa6-Cys 7]-Xbb8-NH2 (Xaa = allo-Thr, D-allo-Thr, D-beta-Hyv, beta-Hyv, D-Thr, and Xbb = Thr or Xaa = Thr and Xbb = allo-Thr, D-allo-Thr, beta-Hyv, D-Thr) contain stereochemical changes in the Thr residues in positions 6 and 8, which allow us to investigate the influence of the stereochemistry within these residues on conformation and binding affinity. The molecular dynamics simulations provide insight into the conformational flexibility of these analogs. The compounds with (S)-configuration at the C(alpha) of residue 6 adopt beta-sheet structures containing a type II' beta-turn with D-Trp in the i+1 position, and these conformations are "folded" about residues 6 and 3. The structures are very similar to those observed for sandostatin, and the disulfide bridge results in a close proximity of the H(alpha) protons of residues 7 and 2, which confirms earlier observations that a disulfide bridge is a good mimic for a cis peptide bond. The compounds with (R)-configuration at the C(alpha) of residue 6 adopt considerably different backbone conformations. The structures observed for these analogs contain either a beta-turn about residue Lys and Xaa6 or a gamma-turn about the Xaa6 residue. These compounds do not exhibit significant binding to the somatostatin receptors, while the compounds with (S) configuration in position 6 bind potently to the sst2, 3, and 5 receptors. The nmr spectra of analogs with (R) or (S) configuration at the C(alpha) of residue 8 are strikingly similar to each other. We have demonstrated that the chemical shifts of protons of residues 3, 4, 5, and 6, which are part of the type II' beta-turn, and especially the effect on the Lys gamma-protons are considerably different in active molecules as compared to inactive analogs. Since the presence of a type II' beta-turn is crucial for the binding to the receptors, the chemical shifts, the amide temperature coefficients of the Thr residue and the medium strength NOE between LysNH and ThrNH can be extremely useful as an initial screening tool to separate the active molecules from inactive analogs.     

Proton n.m.r. investigation of conformational influence of penicillamine residues on the disulfide ring system of opioid receptor selective somatostatin derivatives

Three cyclic disulfide analogs related to somatostatin, D-Phe(1)-cyclo(Cys(2)-Tyr(3)-D-Trp(4)-Lys(5)-Thr(6)-Xxx(7))-Thr(8)- NH2 (where Xxx = L-Pen 1; L-Cys 3; or D-Pen 4) were examined in DMSO-d6 by one- and two-dimensional proton n.m.r. spectroscopy in order to analyze the conformational influence of the position-7 residue on the 20-membered disulfide ring. From these studies it was concluded that all three analogs maintain a beta II' turn solution conformation for the core tetrapeptide -Tyr(3)-D-Trp(4)-Lys(5)-Thr(6)-. However, the disulfide conformation differs in the analogs, with 1 and 3 having a left-handed and 4 a right-handed disulfide chirality.     

Conformational stability and activity of ribonuclease T1 with zero, one, and two intact disulfide bonds

Ribonuclease T1 has two disulfide bonds linking cysteine residues 2-10 and 6-103. We have prepared a derivative of ribonuclease T1 in which one disulfide bond is broken and the cysteine residues carboxymethylated, (2-10)-RCM-T1, and three derivatives in which both disulfides are broken and the cysteine residues reduced, R-T1, carboxamidomethylated, RCAM-T1, or carboxymethylated, RCM-T1. The RNA hydrolyzing activity of these proteins has been measured, and urea and thermal denaturation studies have been used to determine conformational stability. The activity, melting temperature, and conformational stability of the proteins are: ribonuclease T1 (100%, 59.3 degrees C, 10.2 kcal/mol), (2-10)-RCM-T1 (86%, 53.3 degrees C, 6.8 kcal/mol), R-T1 (53%, 27.2 degrees C, 3.0 kcal/mol), RCAM-T1 (43%, 21.2 degrees C, 1.5 kcal/mol), and RCM-T1 (35%, 16.6 degrees C, 0.9 kcal/mol). Thus, the conformational stability is decreased by 3.4 kcal/mol when one disulfide bond is broken and by 7.2-9.3 kcal/mol when both disulfide bonds are broken. It is quite remarkable that RNase T1 can fold and function with both disulfide bonds broken and the cysteine residues carboxymethylated. The large decrease in the stability is due mainly to an increase in the conformational entropy of the unfolded protein which results when the constraints of the disulfide bonds on the flexibility are removed. We propose a new equation for predicting the effect of a cross-link on the conformational entropy of a protein: delta Sconf = -2.1 - (3/2)R 1n n, where n is the number of residues between the side chains which are cross-linked. This equation gives much better agreement with experimental results than other forms of this equation which have been used previously.     

Steric interactions of valines 1, 5, and 7 in [valine 5, D-alanine 8] gramicidin A channels.

When the central valine residues 6, 7, and 8 of gramicidin A (gA) are shifted by one position, the resulting [Val(5), D-Ala(8)]gA forms right-handed channels with a single-channel conductance and average duration somewhat less than gA channels. The reduction in channel duration has been attributed to steric conflict between the side chains of Val(1) and Val(5) in opposing monomers (Koeppe, R. E. II, D. V. Greathouse, A. Jude, G. Saberwal, L. L. Providence, and O. S. Andersen. 1994. J. Biol. Chem. 269:12567-12576). To investigate the orientations and motions of valines in [Val(5), D-Ala(8)]gA, we have incorporated (2)H labels at Val 1, 5, or 7 and recorded (2)H-NMR spectra of oriented and nonoriented samples in hydrated dimyristoylphosphatidylcholine. Spectra of nonoriented samples at 4 degrees C reveal powder patterns that indicate rapid side chain "hopping" for Val(5), and an intermediate rate of hopping for Val(1) and Val(7) that is somewhat slower than in gA. Oriented samples of deuterated Val(1) and Val(7) show large changes in the methyl and C(beta)-(2)H quadrupolar splittings (Deltanu(q)) when Ala(5) in native gA is changed to Val(5). Three or more peaks for the Val(1) methyls with Deltanu(q) values that vary with the echo delay, together with an intermediate spectrum for nonoriented samples at 4 degrees C, suggest unusual side chain dynamics for Val(1) in [Val(5), D-Ala(8)]gA. These results are consistent with a steric conflict that has been introduced between the two opposing monomers. In contrast, the acylation of gA has little influence on the side chain dynamics of Val(1), regardless of the identity of residue 5.     

LncRNA SOX2-OT regulates proliferation and metastasis of nasopharyngeal carcinoma cells through miR-146b-5p/HNRNPA2B1 pathway

Nasopharyngeal carcinoma (NPC) is an aggressive malignancy with a high mortality on account of its frequent metastasis and poor prognosis. An extensive body of investigations has proven that long noncoding RNAs are implicated in a variety of biological processes. Although SOX2-OT has been reported to play an oncogenic role in osteosarcoma, the mechanism of SOX2-OT-driven NPC progression is still obscure. The aim of this study was to elucidate the biological function of SOX2-OT and the related possible mechanism in NPC. In our study, SOX2-OT was notably elevated in NPC samples and cells. Further, a high expression level of SOX2-OT was correlated with poor clinical outcomes of NPC. Results from loss-of-function experiments suggested that knockdown of SOX2-OT repressed cell proliferation, arrested cell cycle, facilitated cell apoptosis, and inhibited cell metastasis of NPC. To further investigate the molecular mechanism of SOX2-OT, miR-146b-5p was found to directly bind to SOX2-OT, which mediated the role of SOX2-OT in NPC tumorigenesis. In addition, HNRNPA2B1 was a target of miR-146b-5p and SOX2-OT modulated the expression of HNRNPA2B1 through competitively binding to miR-146b-5p. At last, we discovered that SOX2-OT regulated NPC progression by targeting miR-146b-5p/HNRNPA2B1 pathway, which may provide more innovative targets for the treatment of patients with NPC.     

A stable dimer in the pH-induced equilibrium unfolding of the homo-hexameric enzyme 4-oxalocrotonate tautomerase (4-OT)

4-Oxalocrotonate tautomerase (4-OT) is a multimeric, bacterial enzyme comprised of 6 identical 62-amino acid subunits, which associate under native conditions to form a homo-hexameric structure stabilized entirely by noncovalent interactions. We have previously shown that the GuHCl-induced equilibrium unfolding of 4-OT at pH 8.5 is well modeled as a two-state process involving only hexamer and unfolded monomer; and we have obtained spectroscopic evidence that intermediate state(s) is (are) populated in the equilibrium unfolding reaction at pHs 6.0 and 7.4 [Silinski, P., Allingham, M. J., and Fitzgerald, M. C. (2001) Biochemistry 40, 4493-4502]. Here, we report on the pH-induced equilibrium unfolding of 4-OT using size-exclusion chromatography (SEC), far-UV-circular dichroism (CD) spectroscopy, and catalytic activity measurements over the pH range from 1.5 to 10.1. Our results indicate that the native hexamer of 4-OT is the predominant species in solution at pHs > or =6.2, that a partially folded dimeric state of 4-OT is stabilized in solution at pH 4.8, and that the enzyme is largely denatured in strongly acidic solutions (pH < or =3.1). GuHCl-induced equilibrium unfolding studies on 4-OT at pH 4.8 indicate that the folded 4-OT dimer populated at this pH is stabilized by 11.7 kcal.mol(-1). The results of biophysical studies on a fluorescent analogue of the enzyme, 4-OT(F50Y), and the results of UV photo-cross-linking studies on a synthetically derived 4-OT analogue, 4-OT(P1Bpa), suggest the polypeptide chains in the 4-OT dimer are nativelike in structure with the exception of their C-termini.     

Guanidine-induced equilibrium unfolding of a homo-hexameric enzyme 4-oxalocrotonate tautomerase (4-OT)

4-Oxalocrotonate tautomerase (4-OT) is a bacterial enzyme that is comprised of 6 identical 62 amino acid subunits. The 4-OT enzyme is an attractive model system in which to study the interrelationship between protein folding, subunit assembly, and catalytic function. Here we report on the GuHCl-induced equilibrium unfolding properties of wild-type 4-OT using catalytic activity measurements and using far-UV circular dichroism (CD) spectroscopy. We demonstrate that the unfolding of wild-type 4-OT in 50 mM phosphate buffers containing 6 M GuHCl is reversible at pHs 6.0, 7.4, and 8.5; and we find that there is both an enzyme concentration dependence and a pH dependence to the equilibrium unfolding properties of 4-OT. Our data suggests that the GuHCl-induced unfolding of 4-OT in 50 mM phosphate buffer at pH 8.5 can be modeled as a two-state process involving folded hexamer and unfolded monomer. On the basis of this model, we determined a free-energy value for the unfolding of 4-OT at pH 8.5 to be 68.7 +/- 3.2 kcal/mol under standard state conditions (1 M hexamer). In 50 mM phosphate buffers at pHs 6.0 and 7.4, only the catalytic activity denaturation curves are consistent with a two-state folding mechanism. At the lower pHs the far-UV-CD transitions are not well described by a two-state model. Our results at pHs 6.0 and 7.4 suggest that intermediate state(s) are populated in the equilibrium unfolding reaction at these lower pHs and that these intermediate state(s) have some helical content but no measurable catalytic activity.     

Synthesis and biological activities of [7-(azetidine-2-carboxylic acid)]-oxytocin and -lysine-vasopressin

[7-(Azetidine-2-carboxylic acid)]-oxytocin and -lysine-vasopressin have been synthesised by a (6 + 3) strategy using protected hexapeptide acids with preformed disulphide bridges, and their biological activities have been investigated. All activities were reduced but not to the same extent. In assays of pressor and antidiuretic activity it was observed consistently that the responses to the vasopressin analogue were of shorter duration than responses to lysine-vasopressin of the same amplitude.     

NMR conformational analyses on (des-bromo) neuropeptide B [1-23] and neuropeptide W [1-23]: the importance of alpha-helices, a cation-pi interaction and a beta-turn

The preferred conformations of the orphan G-protein coupled receptor agonists (des-bromo) neuropeptide B [1-23] and neuropeptide W [1-23], referred to as NPB and NPW, have been determined by (1)H NMR, CD, and molecular modeling. The sequences of NPB and NPW are WYKPAAGHSSYSVGRAAGLLSGL and WYKHVASPRYHTVGRAAGLLMGL, respectively. These are hypothalamic peptides that exert their biological actions on GPR7 and GPR8 receptors. Micellar solutions using the membrane mimetic, sodium dodecylsulphate-d(25) (SDS), were used to mimic a physiological environment for the peptides. The secondary structure of NPB consists of a type II beta-turn involving residues Lys(3) to Ala(6). The C-terminal region of NPB exists in a conformational equilibrium between different secondary structures, including an alpha-helix from residues Arg(15) to Ser(21), and a 3(10)-helix from residues Ser(12) to Ser(21). The N-terminus of NPW exhibits a cation-pi interaction between the Lys(3) side chain and the quadrupole moment of the Trp(1) indole group. At the C-terminus of NPW, a well-defined alpha-helical conformation exists from Arg(15) to Met(21). As NPB and NPW have 91% sequence homology from residues Val(13) to Leu(23), with only residue 21 differing between the two peptides, the similar C-terminal secondary structures of these two peptides are consistent with the sequences. This is supported by the similar CD spectra. The different secondary structures at the N-termini for NPB and NPW point to the importance of the N-terminus in receptor binding. This is consistent with the work of Fujii et al. [J. Biol. Chem. 277, 34010-34016 (2002)] who observed that iodination of the NPB Tyr(2) resulted in decreased agonistic activity at GPR7. In addition, Tanaka et al. [Proc. Natl. Acad. Sci. USA 100, 6251-6256 (2003)] showed that deletion of Trp(1) from NPB or NPW drastically decreased activity at GPR7 for NPB and GPR7 and GPR8 for NPW. Therefore, we postulate that the N-terminus is involved in membrane recognition and receptor binding.     

Influence of N-terminal residue stereochemistry on the prolyl amide geometry and the conformation of 5-tert-butylproline type VI beta-turn mimics.

The effects of N-terminal amino acid stereochemistry on prolyl amide geometry and peptide turn conformation were investigated by coupling both L- and D-amino acids to (2S, 5R)-5-tert-butylproline and L-proline to generate, respectively, N-(acetyl)dipeptide N'-methylamides 1 and 2. Prolyl amide cis- and trans-isomers were, respectively, favored for peptides 1 and 2 as observed by proton NMR spectroscopy in water, DMSO and chloroform. The influence of solvent composition on amide proton chemical shift indicated an intramolecular hydrogen bond between the N'-methylamide proton and the acetamide carbonyl for the major conformer of dipeptides (S)-1, that became less favorable in (R)-1 and 2. The coupling constant (3J(NH,alpha)) values for the cis-isomer of (R)-1 indicated a phi2 dihedral angle value characteristic of a type VIb beta-turn conformation in solution. X-ray crystallographic analysis of N-acetyl-D-leucyl-5-tert-butylproline N'-methylamide (R)-lb showed the prolyl residue in a type VIb beta-turn geometry possessing an amide cis-isomer and psi3-dihedral angle having a value of 157 degrees, which precluded an intramolecular hydrogen bond. Intermolecular hydrogen bonding between the leucyl residues of two turn structures within the unit cell positioned the N-terminal residue in a geometry where their phi2 and psi2 dihedral angle values were not characteristic of an ideal type VIb turn. The circular dichroism spectra of tert-butylprolyl peptides (S)- and (R)-1b were found not to be influenced by changes in solvent composition from water to acetonitrile. The type B spectrum exhibited by (S)-1b has been previously assigned to a type VIa beta-turn conformation [Halab L, Lubell WD. J. Org. Chem. 1999; 64: 3312-3321]. The type C spectrum exhibited by the (R)-lb has previously been associated with type II' beta-turn and alpha-helical conformations in solution and appears now to be also characteristic for a type VIb geometry.